562 N A T U R E S e p t e m b e r 2 9 , 1 9 5 1

100 IQ-io ] 0 - l l f r Concentration (ала./ce.)

Percentage inliibition of germination with respect to control (0-line) : einnamic aldehyde, Д — Д : o-coumaric acid. • — Q ; /З-rt-glucosido-o-coumaric acid, -t (- ; coumarin, О—0,traiis-einnamic acid, x — X ; сгв-cinnamic acid, • — • , tested on the zygotes of the bluatik-os of Vhlamydomonas eugametos. The concentrations for 50 per cent inhibition can be read from the broken

line. + is promotion (above the 0-line)

found i n the case of źro?!S-2-oxy-cninarnic acid (o-coumaric acid). P-tZ-Glucosido-o-ooumaric acid is less active than the aglucone. Coumarin is a hundred times more active ; even at a di lut ion of 10~8 gm./c.c. a statistically significant promotion of germination can be definitely observed. S t i l l more active is trans-cinnamic acid as has been already reported 1 ; though in very dilute solution (10~ 1 0 to 10~1 2 gm./c.c.) no statistically significant promotion of germination was recorded. The range of ac t iv i ty of сгв-cinnamic acid is fifty to eighty times moro than that of the trans form, since even at a di lut ion of 10" 1 1 to 10~1 2 gm./c.c. a significant promotion of germination could bo observed. The three allotropie modifications of cie-cinnamic acid (42° C , 58° C , 68° C.) have the same ac t iv i ty 2 .

The strong act iv i ty of coumarin can be ascribed to the opening of the lactone ring and the formation of сгв-2-oxy-cinnamic acid (coumarinic acid). A s the compound is unstable, its effect could not be tested on zygotes of Chlamydomonas. I t may be assumed that the act iv i ty of fowis-cinnamic acid is duo to the formation of сгв-cinnamic acid during the germination of zygotes, which takes place only i n the presence of l ight 3 . Then i t would be expected that, wi th a sufficiently large number of zygotes, at a di lut ion of 10~1 0 to l f r 1 1 gm./c.c. of trans-cinnamic acid, promotion of germination w i l l be observed. The question of the isolation of natural germination inhibitors from zygotes olChlamydomonae remains st i l l open.

F B A N Z M O E W U S B A S U D E V B A N E R J E E

M a x Planck-Inst i tut fur medizinische Forschung,

Heidelberg. 1 Moewas, Г., Z. Nałurfg., 5 b, 190 (1050). 1 Moewus, F., and Barcrjee, В., Z. Naturfg., в 6 (in the press). 1 Moewas, Г., Biol. Z., 63, 109 (1943).

New Derivatives of Nitroparaffins and their Anti tubercular and Antiricketts ial

Properties A s I have indicated earlier, the reaction of nitro

paraffins wi th formaldehyde and ammonia or amines may lead to the formation of polymers 1 . Fur ther experiments have shown 2 that a number of new ring compounds of 1 : 3-tetrahydro-oxazine (I and

11) and l-oxa-3-aza-c,(/ctooctane (III) type are formed by the interaction of l-nitropropane wi th formaldehyde and ammonia. The products can be transformed into hydrochlorides of the open-chain amines (IV), (V), (VI) or methiodide (VII) . 2-Nitropropane yiolds the amine (VI I I ) 3 wi th formaldehyde and ammonia.

Recently, m y co-workers and I isolated the compounds ( IX) and (X) f r o m nitrocthtaD6* and the compound (XI) from phenylnitromethane 6 , formaldehyde and ammonia reaction products. A l so , an oxazine (XI I ) was prepared from l-nitropropane, formaldehyde and methylamine 6 .

BL N 0 2 C , H 6 N O ,

I О N—R.

NO.,4

C , H / I N — C H , O H

(I) А ^ С . П , , л,=н C H . NO,

di) л,=с,н„ л,=сн, —Чс — сн,он (vu) л,=с,и„ iî^tcH,), те. (XI) Bl=C,H,, Л 2=СН,ОП

(XII) д,=св„ л, = сн,

(III)

С,Н, N O , С,Н В NO.,

/ N H

\ C H , O H

СН,—С—СН,ОН

(V)

/

д. \ /

с н 2 — с

N 0 2

- Rt

N H

(IV) (VIII)

(IX)

СН, — С — Tij / \

R, N 0 2

к . - о н , R . - C H ,

СН 3 N O ,

О N—СН,

СН, N O ,

I N о

к,=011,011 я.=сн, л.=сн,он

(X)

\ / с

/ \ N O , СН.ОН

C H = C H N O ,

0 ' (XIII) (VI)

(XIV) (XV)

(XVI) (XVII)

It, i ' . I u. Я . - С Н , , « . = 0,11,, II, ('li I, /.'s I'Л Is,

fi,=CH..NK, If, = 011 .Oil « , = (Iir2Oil B . - C H , /(, = 011,011

A l l these substances and

NO. 4274 September 29, 1951 N A T U R E 563 Tabic 1 '

Substance I IV V VI VIII IX XII XIII XIV XV XVII Bacteriostatic concentrations (mgm.

per 100 ml.) 7-5-125 7-5-60 7 -5-60 30-64) 15-125 15-30 7-5-30 5-15 30-125 3C-60 30-60

(The wide range of concentrations was produced by examining the action on different strains.) Substances (VII) and (XVI) were found to be inactive.

Table 2

Substance Lethal dose (gin. per kgin. of body-weight

Substance of rats) Intravenous Subcutaneous per os

I 06 1 -5 II — 2-6 (in oil) c. 3-0 III — — c. 3 0 IV 0-2 — V 0-7 2 0

VI 1-1 — VIII 0-3 — IX 0-8 1-2 X — — c. 3-0 XII 0-6 1-8 XIII — 0-15 (in oil) 0-6 XIV — 3-5 XV — 10 XVII 0-3 —

After 35 days, al l survivors were ki l led and the extent of tuberculous involvement was noted and rated. Experiments wi th the substances (I), (V), (VI), (X) and ( X I V ) are being continued.

Substances (XI I ) and ( X V ) were also examined as to their action in vivo against Rickettsia prowazeki. They proved to be more efficient than p-amino-benzoic ac id 9 . Inoculated white mice treated wi th (XI I ) or ( X V ) l ived for nine days ; those treated wi th p-aminobenzoic acid survived four to six days ; controls died after three to four days.

Identification of a Heparin as a Main C o m ponent of the 'Third Factor' involved in

the Virulence-enhancing Act ion of H o g Gastric Mucin

P R E V I O U S w o r k 1 1 has shown that the virulence-enhancing action of hog gastric mucin is due to a synergic action between two non-specific factors (a viscous medium and particulate residue), and a more specific soluble th i rd factor. Par t ia l purification of the latter 3 yielded a heterogeneous product which was predominantly peptide i n nature, but which contained 5-10 per cent of carbohydrato residues. Fur ther purification, using a revised assay 4 i n which the two non-specific factors are maintained constant, has shown that the carbohydrate moiety is responsible for the ac t iv i ty .

After much preliminary fractionation of the crude mucin from fresh hog gastric mucosa?, this moiety was separated into two active fractions by using the different solubilities of their bar ium salts in water and in aqueous alcohol. Frac t ion A is precipitated from aqueous solution by adding 3 per cent barium acetate at p H 7 and standing at 0° for several days. I t has been obtained as an amorphous sodium salt, and examination at p H 4-5 and pH 8-0 by ultra-

Table S'

Daily dose Number of Mortality Substance Xo. of animals (mgm.) Administration days of after 35 Average Average

per mouse administration days tubere. index survival time Streptomycin 15 10 subcut. 80 0 22 35

I 15 10 per os 30 6-6% 44 32-5 11 15 10 30 60-0% 86 31 r.

III 15 10 30 53-3% 72 30-2 V 15 10 20 28-5%

40-0% . 75 32-5

X 15 10 30 28-5% 40-0% . 71 32 0

XII 15 10 28 57-4% 83 311 XIV 15 10 30 33-3% 77 31-2 x v 15 10 30 80-0% 94 31-9

Control 15 — — —— 73-3% 100 28-6

I am much indebted to Prof. S. Ślopek, of the Nilesian Medical Academy, for carrying out the in vitro and in vivo experiments, and D r . J . Venulet , of the Medical Academy, Warsaw, for examining the tox ic i ty of the substances.

T. U R B A Ń S K I Department of Chemistry, Institute of Technology,

Warsaw, and

Institute of Tuberculosis, Warsaw. A p r i l 10.

1 Urbański, T., U.S. Pat. 2,419,043 (1947) ; Brit, Pat. 601,101 (1948). Urbański, T., and Poole, H. J. , Brit. Pat. 616,337 (1949).

' Hirst, B . L., Jones, J. K. N., Minahan, S., Ochyuski, F. W., Thomas. А. T., and Urbański, T., J. Спет. Soc., 924 (1947).

1 Jones, J. K. N., and Urbański, T., J. Chem. Soc., 1766 (1949). 4 Urbański, T., and Lipska, Mrs. В., Roczniki Cïicmii (in tbc press). 5 Urbański, T., and Szczepanik, Mrs. il..Roczniki CAemr,(in the press). * Ochyriski, F. W., Thomas, А. T., and Urbański, T., unpublished work

(Cf. Senkus, U.S. Pat. 2,447,822 ; 1948). Slopek, S., personal communications (January 26 and April 2, 1951).

"Venulet, J. , personal communication (January 26, 1951). 1 Slopek, S., and Eożniecka, Miss D., personal communication

(January 26, 1951).

centrifugation and electrophoresis indicates a single homogeneous substance. A typical batch has an act iv i ty of 3-9 (2-9-5-1) virulence-enhancing units 1 . F rac t ion В is soluble in aqueous solution containing 3 per cent bar ium acetate and is precipitated by adding alcohol to 50 per cent v /v . I t contains no ester sulphate or hexuronic acid detectable by the Disohe oarbazole reaction 5 , and is s t i l l impure, but i t is predominantly carbohydrate, containing a high proportion of hexosamine. A typical batch has an ac t iv i ty of 1-1 (0-7-1-8) virulence-enhancing units 1 . Purification of this compound is being continued.

Ac t ive heparins are mucoit in sulphuric esters i n which the number of sulphate groups can vary con-siderably 6 . Chemical analysis of the essentially homo-geneous fraction .4 clearly indicates that i t is a heparin. This has been further confirmed by comparing the anticoagulation ac t iv i ty of fraction A w i th that of three different, highly active, samples of authentic heparin which were also assayed for virulence-enhancing act iv i ty . I n addition, m y colleague D r . H . P . Lambert bad found that fraction .1 inhibits

image - 0077image - 0078