© 1999 WILEY-LISS, INC.

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that objective in mind. As a consequence, they areeither not in proper focus or with sufficient resolutionto be used in the zoomed-in mode. Of approximately1000 slides of micrographs of three-dimensionalobjects that I use routinely in classroom presentations,perhaps as few as five (5) met the criteria for zoompresentation; new images must be produced. One ofthe most effective presentations of these imagesmakes use of a digital to analog converter, a standardclassroom TV (available routinely in many class-rooms), and any version of Photoshop™. Photoshop™automatically centers the feature selected to magnifyor demagnify. The TV presentation is very effective forimages but ineffective for text. TV presentation isespecially functional as an enhancement for class sizesbelow thirty. A computer projector while also effective(especially for larger classes) is seldom as convenientor available.

BANISH, N., WEISS BIZZOCO, R. L. W., LU, M., ANDSAAVEDRA, S.Department of Biology, San Diego State University, San DiegoCA 92182-0057.

DAPI based discovery of a new group ofextremophiles: hyperthermal acidophilic rods

We report here on the discovery of a new group ofrod shaped hyperthermal acidophiles living in natureat temperatures of 70–80°C at pH 2–3. At pH 2 themaximum temperature at which these organismswere observed was 88°C. That these rods are organ-isms is supported by DNA specific DAPI staining andcombined phase contrast fluorescence microscopy. Inmost acid hot springs examined, these rods were thepredominant organisms above 70°C. They were foundfrom 65–88°C, pH 2–3. Two types of rod shaped cellsexist, one with a cell wall structure that resembles theSulfolobus wall and others that have a multilayeredGram negative type cell wall. 24 hour slide immersionstudies at 65–75°C showed rods were the onlyattached entities, other than sulfur.

ALEXANDER, C.G.Department of Biology, San Francisco State University, SanFrancisco CA 94132.

Parasites as ArtI began photographing parasites when I was work-

ing on shark tapeworms for my doctorate thesis atUCLA in 1953. After I began teaching Parasitology in1955 at San Francisco State I developed a system ofpreparing color-slides using a photomicrographic out-fit obtained with a grant from the National Institutesof Health. I now have several thousand slides of pre-pared and stained specimens of virtually every speciesof parasite from the lab studies of my class and frommy own research. I put together a show using theseslides initially to introduce the students on the firstday of class to a consideration of parasites not as some-thing to be viewed with distaste and horror but as oneintegral part of the whole of “creation,” each with itsown beauty and fascination of complexity. One cannothelp but wonder - why is such intricate design of struc-ture developed simply to grasp and hang on to the vil-lus of a shark’s intestine?

ANTIPA G.A.Department of Biology, San Francisco State University, SanFrancisco CA 94132.

Hi Res/Lo Tech computer imaging for theclassroom

I have found that scanning micrographs, eitherprints or 35mm slides, at reasonably high resolution(i.e., 2–5 Mb/image) provide image files that can be“zoomed-in” during a class session. This allows theopportunity to select parts of an image and magnifyjust that section for the class. The continuity betweenALL low magnification and high magnification imagesadds significantly to clarity for all students, a claritynot generally available in textbooks or standard slidepresentations. That’s the good news. Unfortunately,most existing slides or prints were not produced with

5TH California Microscopy Colloquium

The California State University & Northern California Societyfor Microscopy

Saturday, October 2, 1999

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BARANSKI, M.1, BERDOUGO, E.1,3, SANDLER, J.S.1,3,DARNELL, D. K.2, AND BURRUS, L.W.1,4

1Department of Biology, San Francisco State University, 1600Holloway Avenue, San Francisco CA 941322Department of Biology, Lake Forest College, 555 N. SheridanRoad, Lake Forest, IL 600453These two authors contributed equally to the work.4Corresponding author: LBurrus@sfsu.edu

The dynamic expression pattern of frzb-1suggests multiple roles in chick development

The Wnt family of secreted proteins has been shownto have multiple roles in embryonic development. Frzbproteins have been shown to inhibit Wnt signaling. Inorder to gain a better understanding of the potentialroles of Frzb-1 in chick development, we utilized thepolymerase chain reaction (PCR) to isolate a partialcDNA of the chick orthologue of frzb-1 and compared itsexpression pattern to that of Wnt-1 and Wnt-5a, bywhole mount in situ hybridization. The earliest expres-sion of cfrzb-1 is in cells fated to become neural ecto-derm in streak stage embryos. After primitive streakstages, cfrzb-1 expression is gradually attenuated inthe closing neural tube of the trunk and is concomi-tantly up-regulated in neural crest cells. Finally, cfrzb-1 appears in the condensing mesenchyme of the bonesin both the limb and the trunk. Expression patterns forWnt-1 and Wnt-5a suggest possible antagonistic rela-tionships between cFrzb-1 and these Wnt proteins.

BARLOW, S.B.Electron Microscope Facility, San Diego State University, SanDiego CA 92182-4614.

Education outreach: resources for microscopyin education

Microscopy is a powerful tool for studying and com-prehending structure. It is now possible to operate sev-eral different kinds of microscopes with a desktop com-puter and the appropriate software, regardless ofwhether the microscope is adjacent to that computeror located at a distance. With high-speed Internetaccess and the increasing sophistication of both com-puter hardware and software, students andresearchers can remotely access instruments notowned by their own laboratory. However, simply mak-ing the transmission electron microscope, the scanningelectron microscope, the atomic force microscope, andthe confocal light microscope remotely accessible doesnot insure the incorporation of these tools into the pre-college and college academic curriculum. Several stepscan be taken toward this end. First, instructors need towrite laboratory exercises that can be incorporatedinto student laboratories or daily lesson plans. Theseexercises would emphasize student involvement inplanning and preparing samples to be viewed either inlocal or remotely accessible instruments. Once writ-ten, these exercises would be collated and disseminat-ed. Second, students can use computer simulationsthat mimic digital microscope operation. Third, micro-scopists can make available collections of microscopeimages that can be used as part of the lesson plan.

These three activities are being carried out by theMicroscopy Society of America sub-committee onEducation Outreach. Further descriptions ofmicroscopy outreach resources are available athttp://www.sci.sdsu.edu/emfacility/outreach.html

BART, K.M.1, AND BAILEY, D.G.2

1Department of Biology, Hamilton College, Clinton NY 13323 2Department of Geology, Hamilton College, Clinton NY 13323.

Enhancing undergraduate education throughremote operation of an SEM/ EDS system

At Hamilton College we have tried to broaden anddiversify the pool of students that are able to use theEM facilities. We have done this by providing remoteaccess to the SEM/EDS system in large undergradu-ate courses. Recent advances in digital technologieshave provided new opportunities for dramaticallyincreasing the number of students exposed to SEMand X-ray microanalytical systems. Recently, stu-dents in a 200-level Geology course (Mineralogy)have used the system by remote operation from theirnormal classroom. During class, approximately 25students are able to examine and investigate geolog-ical samples in real time. It is one of the best ways toteach students about the chemistry and structure ofcomplex, polyphase inorganic materials. The hard-ware and software needed to control the system froma remote computer terminal are relatively inexpen-sive and simple to configure. The main requirementsare: 1) a 10 BaseT Ethernet network, 2) computerwith remote access software (e.g. LapLink™); and 3)a data projection system. Use of the EM facilities hasexpanded enormously. Most significantly, many morestudents are now using the instrument for seniorresearch projects. We believe that remote operationof microscopy facilities will become one of the mostefficient and effective ways to teach important scien-tific techniques and concepts to large numbers ofstudents.

BIZZOCO, R.L.W., AND DIAZ, R.G.Molecular Biology Institute, San Diego State University, SanDiego CA 92182.

Effect of carbohydrate on the function of thecontractile vacuole in Chlamydomonasreinhardtii

When Chlamydomonas reinhardtii 406 (mt-), awall-less, flagella-less mutant is incubated in low con-centrations of sorbitol (10 to 70 mM), the time of thecontractile vacuole cycle (filling to emptying) isincreased. We have examined this response using bothphase-contrast and electron microscopy. In normalcontrol cells filling starts when small vesicles fuse intoa large vacuole. Water expulsion begins with a rapidvesiculation of the contractile vacuole. This causes areduction in vacuole volume and an abrupt collapse.During volume reduction water is somehow forced outof the cell at the vacuole-plasma membrane juncture.After sorbitol treatment the contractile vacuole cycletime increases and pulsation of the two vacuoles

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becomes asynchronous, i.e. they fill at the same time.The contractile vacuole in normal control cells has avectoral tendency towards a permanent attachmentsite on the inner plasma membrane surface. Weobserved this vectoral membrane flow during vacuolecollapse both by video microscopy and gluteraldehyde-osmium tetroxide fixed cells. We present micrographsthat depict these chemically fixed cells and cells pre-pared by rapid freezing in liquid propane, followed byfreeze infiltration in osmium tetroxide. Cells exposedto hypertonic medium, such as sorbitol, would ordi-narily be expected to lose water and crenate as a con-sequence of high osmotic strength in the externalmedium. These cells do not crenate. Instead, the con-tractile vacuole behavior becomes abnormal. Althoughit is unclear why the contractile vacuole changes, weoffer a contractile vacuole pumping model.

BLAKE, D.NASA Ames Research Center, Moffett Field CA 94035-1000.

Life on Mars, and life in the Mars meteorite.Has the latter taken on a life of its own?

In 1996, McKay and co-authors announced the dis-covery of evidence of ancient life in a meteorite fromMars. The data consist of five lines of evidence which,while individually weak, according to the authors,together provided compelling evidence of ancient bac-terial life on Mars. Since the original proposal wasmade, three of the lines of evidence have been shownto be either inconclusive, or to have no bearing on life.The two remaining lines of evidence are difficult toeither prove or disprove, but are, in the opinion ofmany researchers, not persuasive. Regardless of theresolution of the Mars meteorite controversy, manybelieve that Mars could have harbored early life.Indeed, the search for evidence of life is one of the cen-tral themes of NASA’s Mars exploration program.

BLINK, A. 1,2, KWONG, A.1,2, ROZENFELD, S.1,LARGMAN, C.1, AND KOMUVES, L.G.2

1Molecular Hematopoiesis Laboratory 2Microscopy and Molecular Histology Laboratory, V. A. MedicalCenter, UCSF, San Francisco CA 94121.

The localization of PRX2, a homeobox gene indeveloping human skin

PRX2 is a homeobox protein thought to regulatenuclear transport of interacting homeobox proteins.Although earlier we established the spatial and tem-poral changes in the expression of a set of homeoboxgenes during fetal skin development, the role of PRX2in skin is not known. The aim of this investigation wasto determine PRX2 localization in developing humanskin. At 10 weeks both the fetal epidermis and perid-erm stained strongly for PRX2. However, at 17 weeksthe basal cells stained only weakly whereas thesuprabasal cells continued to display a strong stainingfor PRX2. Moreover, whereas PRX2 clearly localized tothe cytoplasm of basal cells, both cytoplasmic andnuclear localization was seen in suprabasal cells. Theanlangens of the forming hair follicles and cells in the

dermal papillae did not stain for PRX2. At 21 weeksepidermal staining was similar to that observed at 17weeks. In the fully formed hair follicles strong stainingwas observed in the outer root sheath and the bulge.No staining was seen in the dermal papillae andfibroblasts. In adult skin a decreased cytoplasmicPRX2 staining was seen in the spinous and granularlayers. However, intense staining persisted in the hairfollicles and sweat glands.

BUCHANAN, J., AND SMITH, S.J.Molecular and Cellular Physiology, Stanford University Schoolof Medicine, Stanford CA 94305.

Observations on the fine structure of cellcultures prepared by microwave processing

Microwave processing is a rapid and reliable methodused to prepare monolayer cell cultures for E. M. stud-ies. We have studied hippocampal neurons and MDCKcells by combining confocal and correlative electronmicroscopy. Glass coverslips can be processed in lessthan two hours using conventional fixatives and resinmixtures. The ultrastructure of these cultures is similarto bench processed tissue. Observations on early con-tacts between developing neurons reveal the presence ofsynaptic vesicles and dendritic filopodia.

CHANG, E. AND ANTIPA, G.A.Department of Biology, San Francisco State University, SanFrancisco CA 94132.

Morphogenesis in the ciliate Conchophthirus:structural detail of thigmotactic fieldformation

The ciliature and infraciliature of the thigmotacticfield of C. curtus (Scuticociliatida) is distinctive.Morphogenesis was studied by Antipa andHatzidimitriou (J. Protozool. 28:206-214). Here wereport further study by TEM and provide additionaldevelopmental detail. Within the nine stages of mor-phogenesis previously described by Antipa andHatzidimitriou, we have distinguished two majorphases of thigmotactic field development. Phase 1includes stages 1 through 5 during which the replica-tion zone is formed by duplication of basal bodies with-in all kinetal rows. Monokinetids initially replicate toproduce dikinetids, and then trikinetids, and finallyquadkinetids. This results in a band dense with basalbodies that encircles the cell and demarcates the pre-sumptive proter from the opisthe. All of these newbasal bodies become ciliated. Phase 2 begins at stage6, in which the linear arrangement of the replicationzone basal bodies becomes remodeled. On the left sideof the organism, in the area of the opisthe’s developingthigmotactic field, there is one more round of basalbody proliferation. This produces one additional basalbody for each basal body already present and estab-lishes a zig-zag arrangement of basal bodies charac-teristic of the thigmotactic field. Each new basal bodybecomes ciliated at the end of stage 6, and the thigmo-tactic field of the opisthe is complete. The proter inher-its the parental thigmotactic field.

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CISNEROS-MORENO, Y., AND BIZZOCO, R.L.W.Department of Biology, San Diego State University, San DiegoCA 92182-0057.

Contractile vacuole function inChlamydomonas

This study defines the membrane events in the con-tractile vacuole cycle of Chlamydomonas reinhardtii, asingle cell green alga. It provides a morphological andtemporal basis for the study of membrane fusion andfluid transport across membranes in a cell favorable forgenetic analysis. The contractile vacuole (CV) is anorganelle that serves primarily as a pump to removeexcess water entering the cell by osmosis from dilutesolutions. It has been reported that fluid dischargefrom contractile vacuoles is caused by the systolic pres-sure and that the rate of discharge is higher when thevacuole size at the start of discharge (systole) isgreater. Our study stands in contrast to previously pub-lished work in Chlamydomonas which offers a fusionmodel (CV-plasma membrane) for CV discharge. A lossof both water and CV membrane is postulated to occuras a result of CV-plasma membrane fusion. Our datashow no such loss and that CV membrane persists inthe cytoplasm throughout the pumping cycle. This pro-vides evidence against a fusion mechanism. We alsoshow continuous attachment of the main CV mem-brane to the plasma membrane. No previous study hasshown this. This study defines the different stages ofthe contractile vacuole cycle by correlating observa-tions made by video- and electron microscopy. Theresults we present indicate that the complete cycle con-sists of slow filling and rapid emptying and that the CVmay act as a pump, releasing water through a pore ata plasma membrane attached site.

CONTI, N., AND ANTIPA, G.A.Department of Biology, San Francisco State University, SanFrancisco CA 94132.

Buccal ultrastructure of Conchophthiruscurtus: Implications in the arrangement ofvestibular basal body fibrillar structures

The buccal ultrastructure and infraciliature of C.curtus has been examined by transmission electronmicroscopy. Dikinetal vestibular rows have zigzag cili-ated basal bodies where both have kinetodesmal fiber,transverse fiber, transverse and postciliary microtubu-lar ribbons. Monokinetal vestibular rows have ciliatedbasal bodies each with kinetodesmal fiber, transversefibers, transverse and postciliary microtubular ribbons.Three polykineties contain ciliated basal bodies in threemain kinetal rows with a fourth offset. These basal bod-ies have kinetodesmal and transverse fibers and post-ciliary microtubular ribbons. Haplokinety has zigzagciliated basal bodies with transverse fibers and postcil-iary microtubular ribbons. DKU has monokinetal uncil-iated basal bodies with postciliary microtubular rib-bons. Oral rib ridge has three cytoplasmic layers andmembrane junctions with four and two microtubulesapiece. Cytopharynx has numerous stacked microtubu-lar ribbons along one side. Previous studies show that

basal bodies of the locomotor region are monokinetaland each has a kinetodesmal fiber, transverse fiber,transverse and postciliary microtubular ribbons; thoseof the thigmotactic region are dikinetal, zigzag and haveposterior basal body only with kinetodesmal fiber,transverse fiber and postciliary microtubular ribbons.Comparisons of dikinetal rows with these latter regionssuggest that dikinetal vestibular rows (which aresomatic in origin) probably receive some cellular signalthat makes the dikinetal rows have a characteristicpaired zigzag arrangement and imposes locomotorinfraciliature over both basal bodies.

CORTES, P., AND YOUNGBLOM, J.Department of Biology, California State University, Stanislaus,Turlock CA 95382.

The effect of cigarette tar extract on sisterchromatid exchanges in Leber’s HereditaryOptic Neuropathy

Leber’s Hereditary Optic Neuropathy (LHON) is amaternally inherited disorder that involves variousgenes associated with the electron transport system inthe mitochondria. The primary clinical manifestationassociated with this condition is loss of vision, usuallybilaterally. Several environmental factors have beenimplicated in exacerbating the onset of the symptoms.Two frequently cited factors are excessive cigarettesmoking and alcohol consumption. Despite these spec-ulations, there have been no reported systematic stud-ies to examine the role of these factors in the LHONdisorder. In this study, we investigated the effect ofvarying doses of aqueous cigarette tar (ACT) extracton the level of sister chromatid exchanges in oneLHON patient and one control subject. The sister chro-matid exchange (SCE) assay is a highly sensitivemethod for detecting DNA damage to cells upon expo-sure to various agents. The more damaging the agent,the higher the level of sister chromatid exchanges.Lymphocyte cultures were prepared from whole bloodsamples obtained from the LHON and control individ-uals. The cells were exposed to varying doses of ACTadded at the initiation of the cell cultures. To visualizethe SCEs, the chromosomes were stained with propid-ium iodide and analyzed with a confocal microscope.Using the Mann-Whitney test, the results showed asignificantly higher level of SCEs in the LHON patientfollowing exposure to ACT as compared with the con-trol individual. This study provides the first direct evi-dence that specific components of cigarette smokehave a more deleterious effect on LHON cells thannormal cells.

DEMAREE, R.S., AND FOX, N.E.Department of Biological Sciences, California State University,Chico, Chico CA 95929.

One hour microwave sample preparation forSEM.

The use of microwave-assisted sample preparationfor TEM is slowly gaining acceptance as a routinemethod of tissue processing (Giberson, Demaree andNordhousen, 1997. J. Vet. Diagn. Invest. 9:61-67). We

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have recently reported a one hour microwave enhancedprocedure for preparing bacteria for SEM (Fox andDemaree, 1999. Microsc. Res. Tech. 46:338–339, 1999).We have extended these studies and now report that wecan successfully prepare eukaryotic cells for SEM inunder one hour with microwave enhancement.Fixatives successfully employed include Parducz’ fixa-tive, glutaraldehyde and osmium tetroxide. Eitherethanol or acetone may be used to dehydrate cells.Drying was most rapid using hexamethyldisilazane in aconvection oven, but critical point drying can also beused. We have prepared HeLa cells, human lympho-cytes, Peranema sp., Spirostomum sp., as well as vari-ous plant and animal cells. Examination by field emis-sion SEM at magnifications up to 100,000× revealed nopreparation artifacts.

EVANS, K. L.Department of Biological Science, California State University,Hayward CA 94542.

Using scanning electron microscopy tocharacterize previously unreported freshwatersponges from the California Delta

The California Delta forms an inland coastal envi-ronment and is one of the most extensive bodies ofwater in California. Sponges previously unreportedwere found there year round, mainly growing on thepondweed Egeria but also on the side of certain cov-ered marina floats. Scanning electron microscope(S.E.M.) techniques were used to identify the mostcommon small gray sponges based on structure andasexual reproductive stages. In freshwater spongesone method of asexual reproduction is by gemmules,which have a resistant coat containing unique gem-moscleres made of silica. Such silica spicules inter-fered with additional transmission electronmicroscopy. Gemmosclere rotules (“teeth”) were detect-ed, which numbered fewer than 12 and were 20mm inlength, suggesting the species Ephydatia muelleri(Lieberkuhn). One problem with this identification isthe presence of a microsclere as part of the gemmulecoat; these are not reported to be present in thisspecies. A similar sponge is Spongilla, also reported inmuch of North America. Invertebrates growing overthe sponge surface also were examined, indicating thebryozoan Plumatella, growing within and upon somesponges. The ecological role of such animals is hypoth-esized as patchy but active filters of very small parti-cles, occupying a niche very different from other ani-mals. Keywords: California Delta, freshwater sponges,Ephydatia muelleri, gemmules, gemmoscleres, rotules,bryozoan.

GAERTIG, J.Department of Cellular Biology, University of Georgia, AthensGA 30602-2607.

Molecular and microscopic analysis oforganelle assembly in Tetrahymena

Microtubules form structural frameworks of cellsand organelles which are essential for maintaining

shape and play major roles in cell movement, intracel-lular transport and cell division. Different microtubulesystems (e.g. cytoplasmic network, axonemes, centri-oles, and mitotic spindle) contain MTs that have dis-tinct lengths, spatial arrangement, and dynamics. Ourgoal is to uncover the molecular mechanisms whichregulate the assembly and maintenance of distinctmicrotubular organelles. The ciliated protozoanTetrahymena thermophila maintains at least 17 dis-tinct microtubule structures in a single cytoplasm.Our studies implicate two mechanisms in the assem-bly and maintenance of distinct microtubular systems1) targeting of specific motor proteins to specific micro-tubules and 2) post-translational modifications of asubset of microtubules. We found that kinesin-II, amicrotubular motor protein, is preferentially targetedto cilia and plays an essential role in ciliary assemblyand maintenance. Although disruption of kinesin-IIgenes gave a complex phenotype including paralysis,cell division arrest and lethality, all these changesappear to be induced by the loss of cilia. Distinctmicrotubules are subjected to unique combinations ofseveral types of postranslational modifications. Weshow that polyglycylation, a postranslational modifi-cation specific to cells with axonemes, is essential forsurvival and required for normal functions of multipletypes of microtubules.

GRIFFITH, E.Boston Technology Center, FEI Company, 66A Concord Street,Wilmington MA 01887-2185.

New types of imaging and analytical dataavailable from an ESEM™

In an SEM, the ability to image materials without aconductive coating is the starting point for enhancingthe level of imaging information available from thematerial. Additionally, the ability to image materialsin an SEM in different states and the ability to imagematerials as they change state, provides opportunitiesfor collecting new sample information. TheEnvironmental Scanning Electron Microscope(ESEM™) has a specimen chamber operating modethat allows for a low pressure range of up to 20 torr ofoperator specified gases. These gases can be inert,reducing, or oxidizing. Water vapor is also a gas thatcan be used in ESEM™;. The use of sample stageswith temperature control, in combination with anoperator specified gas, provides for dynamic, in-situ,sample experimentation. Examples of imaging materi-als wet and hot will be explored in this session.

GRIZZI, F.1, INTERNATI, C.M.2, AND DIOGUARDI, N. 3

1Researcher of Scientific Direction, Istituto Clinico Humanitas,Rozzano, Milano, Italy,2Myeloma and Transplantation Research Center, ArkansasCancer Research Center, UAMS, Little Rock, Arkansas,3Scientific Director of Istituto Clinico Humanitas, Rozzano,Milan, Italy; President Centro Medicina Teoretica, University ofStudy, Milan, Italy

Functional and morphological quantitativeevaluation of human myeloma dendritic cells

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Dendritic cells are considered the most importantprofessional antigen presenting cells, distinguished bytheir potency and ability to initiate primary T cellresponses in vitro and in vivo. The most common char-acteristic of these cells is the irregular morphologywhich is strictly linked to their immunological func-tion. Despite the qualitative visual perception of thismorphological complexity, is not available a quantita-tive method for measuring the irregular shape of thesecells for comparing this quality with the specificimmunological function of these cells. In fact, the den-dritiform morphology cannot be correctly measured byconventional Euclidean geometry which is limited toregular structures, practically unknown in nature.Instead, natural forms can be measured by the fractaldimension, which represents the space-filling propertyof an irregular object. In this paper, we describe a frac-tal system, for the quantitative analysis of structuralchanges during dendritic cell differentiation fromblood monocytes, obtained by human myeloma blood.Fractal dimension was compared with the expressionof MHC surface antigens and adhesion/costimulationmolecules. Moreover, our study was undertaken toclarify the morphometric estimations by an imageanalysis system, of various cytological features thatcharacterize these cells. The fractal and morphometricanalyses provided data (p < 0.0005) which suggestthat the early development of irregular dendritiformmorphology reflects high modifications in immunolog-ical properties of the cell. Furthermore, the study ofthe fractal properties of dendritic cells is likely toreveal more about its morphology as well as the kinet-ics of expression of several immunologically importantsurfaces antigens during the monocyte differentiationpathway to dendritic cells.

INGMIRE, P.D., LALLY, D., AND HE, Z.H.Department of Biology, San Francisco State University, SanFrancisco CA 94132.

Tissue and cellular characterization of cellWall-Associated receptor protein Kinases (WAK)in transgenic plants

The plant cell wall plays a dynamic role and func-tions not only as a mechanical structure but as animportant component in signaling pathways as well.The members of a family of transmembrane proteins,called cell Wall-Associated receptor protein Kinases(WAK), are tightly associated with the cell wall andplay a critical role in mediating signals between theextracellular environment and the cytoplasm. The fivemembers of the WAK family exist as a highly conservedgene family clustered together as a 30 kb region onchromosome one in Arabidopsis thaliana. They arecharacterized as having a cytoplasmic serine/threoninekinase which shares high homology among the mem-bers, a single transmembrane spanning region, andextracellular domains unique to each of the membersthat could serve to bind ligands specific for each recep-tor. Genetic manipulation of members of the WAK fam-

ily results in severe morphological alterations in theplant. Transgenic plants show stunted growth,advanced senescence, and necrosis. Microscopic analy-sis of leaves stained with lactophenol trypan blue andexamined using a dissecting microscope confirms celldeath in manipulated transgenic plants. Using scan-ning electron microscopy (SEM), it was also shown thatmanipulated dwarf plants do not show markedly fewercell numbers but rather reduced cell size, suggestingthat disruption of members of the WAK family affectsnormal cell expansion. SEM analysis also shows devel-opmental disruption of trichome formation on youngleaves, and reduced root hair and lateral root formationon affected plants. This work was supported by NIH-MBRS grant 2 S06 GM52588-04 and NIH-RIMI grant(Z.-H. H).

ISAACSON, M.Cornell University, Ithaca NY 14853.

Nanoscopy: A new paradigm for microscopy inthe next century

The last decade has seen a veritable explosion ofnew microscopies aimed at the exploration of innerspace. In the past however, the tools used for thisexploration were large scale complex instruments,scanning transmission and scanning electron micro-scopes for example. In fact, the smaller the object to beexplored, the larger and more complex was the instru-ment. This has changed with advent of scanned tipinstruments; not necessarily the complexity, but thesize scale of the “interrogator.” As we look at the nextcentury there will be this paradigm shift in how we goabout the exploration of inner space. No longer will theobserving tools be orders of magnitude larger than theobject to be viewed, but rather on the same order ofmagnitude in size. This shift has occurred largely thruthe ability to use large scale tools to create small scaledevices. In this talk, we will explore this paradigmshift, and review the micro observational tools we haveat hand and predict what we might have as we beginthe new millennium.

KOMUVES, L. G.Microscopy and Molecular Histology Laboratory, University ofCalifornia, San Francisco, V. A. Medical Center (190), SanFrancisco CA 94121.

Activation of peroxisome proliferator-activatedreceptor-α regulates keratinocytedifferentiation

Ligands of peroxisome proliferator-activatedreceptor-α (PPARα) stimulate keratinocyte differen-tiation and accelerate epidermal development in fetalrat skin explants (Komuves et al., J. Invest.Dermatol. 1998, 111: 429-433). The aim of the presentstudy was to determine if topically applied PPARαligands (clofibrate and Wy-14,643) could regulatekeratinocyte differentiation in adult epidermis.Topical treatment with PPARα activators resulted ina decreased epidermal thickness. The expression of

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structural proteins of spinous and granular layers(involucrin, filaggrin, loricrin) increased followingtopical treatment with PPARα activators. This treat-ment also decreased cell proliferation and increasedapoptosis in the epidermis. Furthermore, topicaltreatment with PPARα activators resulted in rapidnormalization of both subacute (induced by repeatedbarrier abrogation) and chronic (caused by essentialfatty acid deficiency) hyperproliferative skin condi-tions. Topical treatment with PPARα activatorsresulted in a decrease in epidermal hyperplasia inboth models of hyperproliferation. Following topicalclofibrate treatment, PCNA-expressing cells wererestricted to the basal layer, similar to normal epi-dermis. In hyperproliferative epidermis the expres-sion of involucrin, filaggrin, and loricrin wasdecreased. Following topical treatment with PPARαactivators staining for these mRNAs and proteinsincreased towards normal levels. Finally, topicallyapplied clofibrate also increased apoptosis. The pre-sent study demonstrates that topical PPARα activa-tors have profound effects on gene expression, cellproliferation and apoptosis both in normal andhyperproliferative epidermis. This study identifiesPPARα activators as novel potential skin therapeuticagents.

KWONG, A.1,2, ROZENFELD, S.1, LARGMAN, C.1, ANDKOMUVES, L. G.2

1Molecular Hematopoiesis Laboratory 2Microscopy and Molecular Histology Laboratory, V. A. MedicalCenter, UCSF, San Francisco CA 94121.

Immunohistochemical localization of HoxB13during fetal mouse development

Homeobox genes regulate embryonic develop-ment, including organogenesis. To understand therole of HoxB13 in mouse development (E13-E17) weanalyzed HoxB13 localization by immunohistochem-istry. Strong HoxB13 staining was seen in fetal epi-dermis but not in hair follicles and dermis.Interestingly, the stratified keratinizing epitheliumof the tongue did not stain for HoxB13 whereasstrong staining was seen in the oral mucosa. NoHoxB13 staining was found in developing teeth.Striated muscle cells, including skeletal muscles, thediaphragm, the musculature of the tongue, heartmuscles stained strongly for HoxB13 whereas nostaining was seen in smooth muscle. The liverparenchyma did not stain for HoxB13 during E13-15, but was weakly positive later. The thymus andhaemopoietic tissues (liver and bone marrow) didnot stain for HoxB13. Similarly, no staining was seenin the pancreas and in the salivary glands. In thelung HoxB13 staining was restricted to the airways,no staining was seen in the respiratory epithelium.Weak staining was found in the collecting ducts inkidney. No HoxB13 staining was observed in theintestinal tract (stomach, small intestine, largeintestine). Whereas the ependymal cells in the

choroid plexus displayed intense HoxB13 staining,no staining was seen in the central nervous system(brain and spinal cord). Therefore, HoxB13 has com-plex expression pattern during organogenesis infetal mouse. It is mainly expressed in organs of ecto-dermal and mesodermal origin (such as epidemis,striated muscles), whereas it is not present in organsoriginating from the endoderm (such as the intesti-nal tract and digestive glands).

LAU, B.1,2,3, KWONG, A.1,2, LARGMAN, C.1, ANDKOMUVES, L.G.2

1Molecular Hematopoiesis Laboratory, V. A. Medical Center,UCSF, San Francisco CA 94121 2Microscopy and Molecular Histology Laboratory, V. A. MedicalCenter, UCSF, San Francisco CA 94121.3B. Lau (A. Lincoln High School) was supported by the UCSFScience and Health Education Partnership Summer InternshipProgram.

Double immunofluorescent staining withtyramide-mediated signal amplification usingmouse monoclonal antibodies

Fluorescence-based immunohistochemical detec-tion methods are essential in double-labeling studieswhere information about the spatial relationshipbetween different proteins is sought. The feasibility ofdouble-labeling experiments, however, is often compro-mised by the limited availability of primary antibodiesraised in different species. Our goal was to determinewhether mouse monoclonal antibodies could be usedfor double immunofluorescence with tyramide-mediat-ed signal amplification (TSA). To establish double-labeling TSA on paraffin-embedded sections, we usedmonoclonal antibodies against human keratin 14 andfilaggrin. These proteins were specifically detected insingle-labeling TSA, using an HRP-conjugated anti-mouse IgG as a bridge reagent. In the double-labelingexperiments the antigens were detected sequentially,with FITC- or Cy3-labeled tyramide reagents. A set ofcontrols established that TSA during the localizationof the first antigen did not interfere with TSA detec-tion of the second antigen. Moreover, TSA preventedthe non-specific deposition of the bridge antibody tothe sections during the detection of the second anti-gen. Using this technique we were able to identify anovel cell in dysplastic epidermis of basal cell carcino-mas. These solitary cells are wedged between basalcells and possess an unusual phenotype, expressingboth early (keratin 14) and late markers (filaggrin) ofkeratinocyte differentiation.

LAUZON, C.R., AND POTTER, S. .Department of Biological Sciences, California State University,Hayward, Hayward CA 94546.Nitrogen utilization by bacteria in the gut oftwo tephritid species.

Tephritid flies consume a variety of microorganismswhile feeding on natural food sources such as birdfeces, plant structures, and aphid honeydew. Despitethe consumption of an array of microbial species, onlytwo bacterial species are consistently recovered from

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their intestines. This selection suggests that these bac-teria contribute to the welfare of the insects. Althoughthe exact mechanism(s) for selection of these bacterialspecies remains to be defined, we present evidencethat the bacteria participate jointly in nitrogen cyclingfor the fly using biochemical assays alone and coupledwith electron microscopy.

LIM, J.J., AND KOCH, R.A.Department of Biological Science, California State University,Fullerton, Fullerton CA 92834.

Integrins and cadherins as cell adhesionmolecules in ascidian sperm mitochondrialtranslocation.

Mitochondrial translocation in the sea squirt,Ascidia ceratodes, is the movement of the sperm mito-chondrion from the head to tail during fertilizationand is an actin:myosin- dependent process that definessperm activation and provides the force for penetra-tion (Lambert & Lambert, Dev. Biol. 106:307, 1984).Activation of myosin filament formation has been pre-viously supported by the work of Koch et al. (Dev. Biol.,submitted). Actin polymerization, a process not wellstudied in ascidian sperm, requires the appropriateactin binding proteins and multi-protein complexesthat anchor species-selective cell adhesion moleculesto the sperm cell plasma membrane for stability andinitiation of signal transduction. These cell adhesionmolecules mediate the association of actin with eggsurface anchor proteins to form multi-protein com-plexes that recruit signaling molecules and activateactin-myosin dependent motility (Porter & Hogg,Trends in Cell Biol., 8:10, 1998). I hypothesize thatintegrins, a family of transmembrane glycoproteinsthat consist of noncovalent heterodimers, are presentin A. ceratodes sperm and are responsible for anchor-ing and initiating mitochondrial translocation duringfertilization. However, integrins have not yet beenidentified in the ascidian sperm. To test this hypothe-sis, I plan to use fluorescently tagged and colloidalgold tagged antibodies to detect the presence of inte-grin in A. ceratodes sperm using light and electronmicroscopy. Preliminary evidence at the light micro-scope level using anti-integrin monoclonal antibodies(Transduction Labs, #141720) supports the hypothe-sis. The alternative hypothesis that adhesion is viacadherin molecules is currently being tested. Electronmicroscopy will follow. (Supported by CSUF Biology;NIH R15HD36500.)

MATEY, V.E., DEVAUX, C.A., AND WALSH, T.A.Department of Biology, San Diego State University, San DiegoCA 92182.

Surface ultrastructure of parasitic nematoda -Physaloptera turgida

For the first time, SEM was used to study key taxo-nomic characters of Physaloptera turgida Rud., 1819(Nematoda: Spiruroidaea) from the opossumsDidelphis virginiana. 10 male and 10 female wormsfrom stomach of 5 infected animals trapped in SanDiego County were examined. Cephalic end of P. turgi-

da is similar in both sexes and formed by two largelips. Each lip is armed with tripartite tooth, externaltooth and numerous lateral teeth. Two knob-like papil-lae, anterior sensory amphid, and three sensitiveporous structures are placed on the lip surface. Dilatedcuticle surrounding cephalic end forms the collarettewith two papillae. Posterior end of male and femaleworms is different. Male: caudal alae is well developedand supported by four pairs of pedunculated papillae.Cloaca is located in the upper part of tail, number ofspicules varies. Two spicules were found in four males,one spicule in three males and none in two males.Seven button-like papillae are associated with cloaca,another three pairs are situated below it. Posteriorreceptors, phasmids, are placed before the second pairof papillae. Ventral surface of the male tail showsthree different patterns of ornamentation specific forthis parasite. Female: vulva is located in the anteriorpart of body. Conical posterior end bears crescent analopening and few phasmids.

MILLER, J.J., AND ANTIPA, G.A.San Francisco State University, San Francisco CA 94132.

Ultrastructure of Fernandez-Galiano stainedConchophthirus curtus

The Fernandez-Galiano ammoniacal silver carbon-ate method is frequently used for viewing themacronucleus and the kinetal rows of ciliates bymeans of light microscopy. Light micrographs ofConchophthirus curtus indicate that ciliary tips, basalbodies, kinetodesmal fibers, and the macronucleus arestained. The DKU (a putative group of submergedbasal bodies) does not appear to stain. This could bedue to differences in the fiber system of the DKU whencompared to other kinetal rows. Transmission electronmicrographs confirm that ciliary tips, basal bodies(particularly the transition zone and the cartwheelregion), kinetodesmal fibers, and the macronucleus arestained. In addition, the transverse fiber appears topick up the stain. Staining of the transverse fiber andthe ciliary tips is a novel finding. It is possible thatsome feature of the cilium confers thigmotactic abilityto C. curtus which is also responsible for the argen-tophilicity of the tips. However, cilia in all areas of thecell stained in the same way. Could it be that all of thecilia of C. curtus are potentially thigmotactic and thedensity of cilia in the thigmotactic region as comparedto the other regions makes this region sticky?

MORGAN, C.L.1, LAUZON, C.R.2, AND SMITH, N. R.2

1Department of Mathematics and Computer Science;2Department of Biological Sciences, California State University,Hayward CA 94542.

Development of a standard model for remotecontrol of scientific instrumentation

Faculty and staff of the Microscope and GraphicImaging Center (MAGIC) at California StateUniversity, Hayward (CSUH) have been involved forseveral years in developing software for a standard-ized architecture for remote control of scientific instru-mentation. Commercial software and freeware are

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available for connecting computers to control and com-municate with scientific instruments, but they do notafford the flexibility, the security, and the scalabilitythat custom software can offer. When we acquired ourPhilips (FEI) XL-40 scanning electron microscope, thechallenge to develop a software system to gain remoteusers a standardized Internet-accessible system wasundertaken. Using the remote control software devel-oped by faculty and students in the Department ofMathematics and Computer Science, the SEM hasbeen controlled locally, nationally and internationally.The SEM has been used by sister CSU campuses, localcommunity colleges and high schools, and through in-home use by staff and faculty. An adjunct projectinvolving HPLC remote instrumentation control incor-porates the same basic computer-system architecturaldesign. This model system can be used to control anydigital visual-output instrumentation.

MORIMUNE, M.1, 2, 3, KWONG, A.2, ROZENFELD, S.1,LARGMAN, C.1, AND KOMUVES, L.G.2

1Molecular Hematopoiesis Laboratory 2Microscopy and Molecular Histology Laboratory, V. A. MedicalCenter, UCSF, San Francisco CA 94121.3M. Morimune (A. Lincoln High School) was supported by UCSFScience and Health Education Partnership Summer InternshipProgram.

Immunohistochemical localization of HOXB4during fetal human skin development

Our previous studies have established the spatialand temporal changes in the expression of severalhomeobox genes during fetal skin development(Differentiation 62:33-41, 1997; J. Invest. Dermatol.110:110-115, 1998). To expand these studies, wereport here the developmental changes in HOXB4expression in fetal human skin. Whereas at 10weeks only weak staining was seen in the epidermis,at 17 weeks the basal cells strongly stained forHOXB4. Moreover, several cells stained in the core ofthe forming hair follicles. No staining was seen inthe dermal papillae. At 21 weeks, in the stratifiedepidermis the basal cells (which are proliferatingcells) stained strongly for HOXB4. Moderate stain-ing was also observed in the suprabasal cells. In thefully formed hair follicles the cells in the outer rootsheath, in the bulge (where the epidermal stem cellsare located), and in sebaceous glands stained forHOXB4. However, no staining was seen in the der-mal papillae and fibroblasts. This expression pattern(presence of HOXB4 in proliferating cells and instem cells) suggests that HOXB4 plays a role in theregulation of cell proliferation and/or stem cellrenewal in developing fetal human epidermis.

MURPHY, J. A.San Joaquin Delta College, Microscopy Technology Center, 5151Pacific Ave., Stockton CA 95207. jmurphy@sjdccd.cc.ca.us.

The Challenges of Teaching ResearchMicroscopy at a Community College

San Joaquin Delta College has a unique 2 yrMicroscopy Certificate Program which successfullyplaces 95% of its students either in the job market

directly or as transfers to other 4 yr institutions.Microscopy, in general, is research oriented in that itprovides tools to solve problems. The program hasadvanced project courses which train the student to doa research project from the very beginning stages ofexperimental design through data collection andanalysis. There are however, many challenges forteaching research microscopy at the community col-lege level, e.g., library resources, how class logisticsaffect experiments, equipment necessary, cost, staff,and space required. Although research is not the mis-sion of most Community Colleges, in order to providehigh technology skills required in today’s job market,they need to address the elements of scientific method,e.g., critical thinking, problem solving, organization,and team work. Instrument based programs needmore financial and space support than traditional pro-grams and absolutely need District andAdministrative support at both the local and statelevel minimally. In addition, funding agencies need tore-think how they distribute funds, i.e., funding ofresearch training activities such as for supplies,instrumentation, and buildings, regardless if it is a 2yr, 4 yr, or graduate institution. We must meet thesechallenges in innovative ways as in the end, it allreflects on our society’s commitment to our young peo-ple, to make them the best that they can be. They are,after all, our future.

PARVIN, B.1, TAYLOR, J.1, AND O’KEEFE, M.A.2

1ICSD, Lawrence Berkeley Laboratory, Berkeley CA 94720 2MSD, Lawrence Berkeley Laboratory, Berkeley CA 94720.

Deepview: A channel for distributedmicroscopy.

The Materials Microcharacterization Collaboratory(MMC), a pilot project of DOE2000, unites DOE userfacilities at LBNL, ANL, ORNL, the University ofIllinois and NIST. The MMC offers materials scientistsaccess to an on-line virtual laboratory that collec-tively houses the nation’s most advanced electronmicroscopes, expert microscopists, and othermicrocharacterization tools. As an integral part ofthe MMC, we have developed a generalized“Microscopy Channel” over the wide area network. Amicroscopy channel advertises a listing of availableonline microscopes, where users can seamlessly par-ticipate in an experiment, acquire expert opinions,collect and process data, and store this informationin their electronic notebooks. This channel is a col-laborative problem-solving environment (CPSE)that allows for both synchronous and asynchronouscollaboration. Deepview, provides a layer of abstrac-tion for controlling any type of microscope, a commonset of utilities for information management andtransaction, and the analytical capabilities neededfor online microscopy. With Deepview, we have pro-vided remote users with collaborative access to elec-tron microscopes in Berkeley (LBNL) and Tennessee(ORNL) from several materials science laboratoriessited at locations distributed across the country.

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POTTER, S.E., AND LAUZON, C.R.Department of Biological Sciences, California State University,Hayward, Hayward CA 94542.

Bacterial assemblages in the gut of twotephritid species.

The true fruit flies are destructive insect pests of eco-nomically-important agricultural crops such as coffee,citrus, and nuts. Current control methods for theseinsects include the use of pesticides that affect nontar-get species, pollute the environment, and are expensive.These are incentives for researchers to find new controlmethods that are species specific. In order to achievethis, the biology of the insect must be more completelyunderstood. We have looked closely at the bacteria thatreside in the intestines of two tephritid pests. It hasbeen hypothesized that bacteria are important in thelife cycle of these insects. We have found a close associ-ation exists between the insect gut tissue and bacteria.Bacteria attach to the peritrophic matrix and form abiofilm. The biofilm includes packets of bacteria, anarchitecture conducive to the flow of nutrients in andthe excretion of waste products out of the gut. Thesedata provide evidence that physical factors may beinvolved in selection of bacteria within the fly gut.

SADREDDIN, A., LUONG, J., LEE, R., CARMEN, G.,AND BRECKLER, J.Department of Biology, San Francisco State University, SanFrancisco CA 94132.Immunolocalization of cytoskeletal elements inretinal pigment epithelium (RPE) duringphagocytosis

RPE are phagocytic cells in the vertebrate eye thatengulf rod outer segments (ROS) that are shed withlight on-set. Phagocytosis is an actin and microtubule-dependent process. Therefore we are studying actinand microtubule cytoskeleton during RPE phagocyto-sis. We are particularly interested in ADF (actindepolymerizing factor). ADF depolymerizes actin fila-ments in the cytoskeleton and allows them to performand rearrange for phagocytosis. Immunocytochemistryis used to study the distribution of ADF using anti-bodies and secondary fluorescent detection methods.In addition, we are using Rhodamine-Phalloidin stain-ing method to localize the F-actin in the cell and com-pare this localization pattern to the ADF pattern. Wehope to answer the following questions: Where is ADFmore concentrated, and does it localize in areas wherephagocytosis is taking place? Does ADF co-localizewith F-actin? In order to follow microtubules duringphagocytosis, we are using anti-tubulin. We also planto use anti-myosin VI to see if this particular type ofmyosin is involved. Two sets of experiments are inprogress. One is on Sunfish and the other is on albino(unpigmented) rat eyes. In order to look at the pres-ence of ADF in cells that are actively phagocytizing, weexamined different time points. We performed rat eyedissection one hour after light on-set (8 am), at whichthere is a burst of ROS shedding and most of phagocy-tosis is taking place. Another experiment is the late

afternoon dissection (approximately 8 hrs. after lighton-set). In our study, we are comparing and contrast-ing these time points in order to determine any shift indistribution of ADF, actin, microtubules and myosin VIduring several stages of engulfment in vitro.

SMITH, N.R.Microscope and Graphic Imaging Center (MAGIC), CaliforniaState University, Hayward CA 94542.Teaching Microscopy at CSUH: Then and Now

Electron microscopy classes were first taught atCSUH in 1971. The classes were small in size with amaximum of six students for the transmission electronmicroscopy course and as many as 14 for the scanningmicroscopy course. Then, as now, students wererequired to produce a finished project at the end of eachterm. However, now rather than spending many hoursin the darkroom, students are producing images ontheir computers. The development of remote microscopyhas changed our perspective on how microscopy coursesare taught and has reduced the limitations on class size.The one instructor, one student approach to teachingmicroscopy is elevated to another level and it can nowbe done in a lighted room, thanks to digital microscopy.Using telemicroscopy methods we can bring images intoclassrooms on our campus and outreach to local com-munity colleges and high schools through the Internetthus serving many more students than in the past.More students can be exposed to the direct experienceof all types of digital microscopy. Digital output onTEMs and SEMs has saved students a tremendousamount of time in producing micrographs and hasreduced costs to the facilities. Students are able to saveimages on disks and output them from their homes orschools and incorporate them into imaging enhancingand analytical software.

THOMPSON, J.M.Biology Department, California State University, SanBernardino CA 92407.Remote Microscopy - current status and futurepromise

Remote microscopy can take several forms. On a sim-ple level, it is nothing more than sending a micrographto a colleague. This is a technology which has existed foryears, but which limits the immediate interaction andvalue to both participants. New technologies now allowfor real-time interaction between microscopists and col-leagues who are at remote, often distant, sites. Thisinteraction can take the form of person to person com-munication using videoconferencing hardware and soft-ware through internet connections, and multiple remoteobservers can also participate with the appropriate soft-ware. If the microscope can be controlled by a computer,the interaction may also occur by remote control of themicroscope by the colleague at the remote site.Limitations in existing hardware and software includeslow rates of internet transmission, low resolutionimages, small screen size of images, and often a lack ofaudio capabilities. In this symposium, we will discussthe hardware and software currently available, as well

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as future developments to enable these remote interac-tions. Demonstrations of current remote microscopyfrom several sites will also be presented.

TIFFANY, M.A.Department of Biology, San Diego State University. San DiegoCA 92128.Studies on the skeletal development ofHermesinum adriaticum Zacharias, a flagellatefrom the Salton Sea, California.

Hermesinum adriaticum is a rarely reported unicel-lular biflagellated organism of uncertain classifica-tion. Unlike silicoflagellates it has a solid internalsiliceous skeleton. It was found in cyanobacterial matsand in the sediments of the Salton Sea, a salt lake inCalifornia, USA. Stages of the developing skeletonwere studied with SEM and the progression fromsmall tetraxial daughter skeletons to complete asym-metrical adult skeletons is presented.

VANDIVER, A. AND MENON, J.Department of Biology, William Paterson University of NJ,Wayne NJ 07470.Retinoic acid and apoptosis

The thyroid hormone-induced tissue remodelingthat occurs during metamorphosis is generally accom-plished by the death of larval cells (via apoptosis) andthe growth and differentiation of stem cells into anadult organ. Retinoids (include retinol, retinal, transand cis form of retinoic acid) have been linked to theinduction of apoptosis in several in vivo and in vitromodels. Thyroid hormone receptors and retinoic acidreceptors belong to the same family of nuclear recep-tors. In Xenopus laevis it is shown that thyroid hor-mone receptors (TRs) and retinoic acid X receptors(RXRs) function together in vivo to regulate T3response genes that are normally expressed duringmetamorphosis. Whole tails of Xenopus laevis weretreated in vivo to regulate T3 response genes that arenormally expressed during metamorphosis. Wholetails of Xenopus laevis were treated in vitro withretinol palmitate (2IU/ml). The tail epidermis wasprocessed for scanning and transmission electronmicroscopy. Under SEM, retinol treated tail epidermisshowed a loss of intercellular connections as well asmicrovilli at their apical surface. Also there was pit-ting on epidermal surface of retinol treated tail. Incontrast, microvilli were clearly visible in epidermalcells was observed following retinol treatment. Musclefibers were also affected: muscle bundles were lessnumerous and more degenerated in retinol treated tis-sue than in the control. Role of retinoids in inducingapoptosis will be discussed.

WRIGHTING, D., AGNEW, B. AND BRECKLER, J.Department of Biology San Francisco State University, SanFrancisco CA 94132.Involvement of the actin cytoskeleton in thephagocytosis of rod outer segment (ROS) disksby retinal pigment epithelial (RPE) cells

The Retinal Pigment Epithelium (RPE) consists ofsupportive cells beneath the vertebrate retina. One of

the primary functions of the RPE is to phagocytoserod outer segment (ROS) disks shed daily from thedistal tips of the photoreceptors. The function of RPEphagocytosis is critical to retinal integrity, adhesionand survival. Striking evidence has been collected forthe role of actin cytoskeletal elements during theprocess of phagocytosis and specifically duringphagocytic cup formation. The initial purpose of ourstudy is to directly visualize the dynamics of rapidactin filament turnover during RPE phagocytosis.Once G-actin addition involved in cup formation innormal cells has been characterized, we will studythe specific roles of actin depolymerizing factor(ADF) in this process by transfecting cells with aden-oviral vectors containing mutant ADF isoforms. Inour studies, the use of the RPE-J cells, an immortal-ized RPE rat cell line, has proved invaluable. TheRPE-J cells possess the important aspects of RPEmorphology and function, including the ability to rec-ognize and phagocytose ROS in vitro. We have devel-oped an assay that incorporates exogenous fluores-cent and biotinylated G-actin into RPE-J cells tovisualize actin rearrangements during phagocytosisby both light and electron microscopy. Support forthis research provided by NIH-RIMI #RR11805 to JBand NIH-BRIDGES to DW.

YOUNGBLOM, J.H., WILKINSON, J., ANDYOUNGBLOM, J.J./Department of Biology, California State University, Stanislaus,Turlock CA 95382.Remote access confocal microscopy.

In recent years, there has been a growing interestin the development of remote access capabilities to avariety of microscopy systems. While certain types ofmicroscopes, such as electron microscopes and scan-ning probe microscopes have been well established fortelepresence microscopy, there have been no reports ofsimilar developments for the confocal microscope. AtCalifornia State University-Stanislaus, home of theCSUPERB (California State University Program forEducation and Research in Biotechnology) ConfocalMicroscope Core Facility, we have established a remoteaccess confocal laser scanning microscope facility thatallows users with virtually any type of computer plat-form to connect to our system. Our Leica TCS NT con-focal system, with an interchangeable upright andinverted microscope set up, is accessible to any autho-rized user via the Internet by using a free softwareprogram called VNC (Virtual Network Computing).Once connectivity is established, users are able to con-trol virtually all the functions to conduct real timeimage analysis and quantitative assessments of theirspecimen. The ease of connectivity to the microscopesystem and the relatively rapid speed of image trans-fer allow accessibility of this highly sophisticatedinstrument to the larger global community. A sharedresource facility of this type, without geographic con-straints, helps to catalyze the interaction of individu-als with diverse interests and expertise, and promotesa center for academic collaboration and innovation.