Louw, H.A.

Scheffler, J.G.

English

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6 August 1966 S.A. MEDICAL JOURNAL 701EXPERIMENTAL BIOLOGY GROUP: SUMMARIFS OF SCIENTIFIC PAPERSAbstracts of papers read at the 20th Scientific Meeting of the Experimental Biology Group (EBG):THE GEL IMMUNE DIFFUSION TECHNIQUE AS A METHOD FOR STUDYING THE ANTIGENICRELATIONSHIPS AMONG RHIZOBIAJ. G. SCHEFFLER* AND H. A. Louw, Department ot Microbiology, University ot StellenboschThe gel immune diffusion technique has not been widely usedfor studying the serology of rhizobia It was therefore thoughtof interest to compare the results obtained by this methodwith those of the classical agglutination technique, performedas described by Loos and Louw.' Antisera were preparedagainst a number of rhizobial isolates, isolated from uninocu-. lated clovers collected in the Stellenbosch district.The antigen suspensions used in the gel immune diffusiontests were washed, using 0·85% saline, and concentrated bytwo successive centrifugations (10,000 r.p.m. for 10 min.). Thepellets were resuspended in small volumes of saline to obtainheavy cell suspensions which were standardized to a cell con-tent of approximately 100 mg. dry weight per m!. (dried at80°C to constant weight).The gel immune diffusion plates were prepared in petri-dishes (diameter 9 cm.) using 0·75% agar, 0·85% saline and0·025% sodium azide. The plates were poured using 20 ml. ofagar. Wells were cut in a hexagonal pattern around a centralwell. The wells had a diameter of 7 mm. and were spaced4 mm. apart. Undiluted serum was placed in the central welland antigen suspensions in the surrounding wells. The preci-pitation bands were recorded after 3 days at room tempera-ture (Dec.-Jan. 1965, aver. approx. 20°C) in a glass containerhaving a moist atmosphere. Whole cells were used as antigensin this study, since Dudman' found that concentrated suspen-sions of washed whole cells of rhizobia gave rise to the samenumber and pattern of precipitation bands as concentrated so-lutions of extracellular diffusible antigens. The medium used*Present Address: Faculty of Agriculture. University of Pretoria.for obtaining the antigens was also tested against all theantisera, but HO bands were formed.It was found that the isolates always produced strong, widebands when tested against their homologous antisera Strong,wide bands were sometimes also formed when isolates weretested against sera prepared against other organisms. The pro-duction of strong identical bands was always correlated withidentical somatic agglutinating properties. This was not so inthe case of the flagellar agglutinating properties.Isolates which showed strong somatic agglutination (at dilu-tions higher than 1/200) were always found to share extra-cellular diffusible antigens with the antiserum-producingantigen. When weak agglutination occurred, however, commonextracellular diffusible antigens were not necessarily found.Agglutination sometimes occurred even though the isolatesshared only a single extracellular diffusible antigen, while inother cases no agglutination occurred although the isolatesshared as many as 3 bands.It can be concluded that when using only the homologousantiserum to identify a particular rhizobial strain among rhizo-bial isolates, the gel immune diffusion technique is a muchfaster method than the agglutination technique. The identifica-tion is not absolute, however, since the results obtained bymeans of the gel immune diffusion technique are not alwayscorrelated with the flagellar agglutinating properties. However,the somatic antigens have been shown by Vincent'" to behighly strain specific.1. Dudman. W. F. (1964): J. Bact., 88, 782.2. Loos, M. A. and Louw, H. A. (1964): S. Afr. J. Agric. Sei., 7, 135.3. Vincent, J. M. (1941): Proc. Linn. Soc. N.S.W., 66, 145.4. Idem (1942): Ibid., 67, 82.OXIDATIVE PHOSPHORYLATION IN HEREDITARY CARDIOMYOPATHYA. LOCHNER, CSIR Degenerative Diseases Group, University ot Stellenbosch Medical School, Karl Bremer HospitalThe oxidative phosphorylation process in hereditary cardio- studies to determine where in the electron transport chain un-myopathy was studied in the Syrian hamster, Mesocricetus coupling occurs, indicated that the phosphorylation in theauratus. These animals spontaneously develop a muscle dys- cytochrome oxidase region was significantly depressed.trophy together with cardiomyopathy which becomes progres- These results are in concomittance with those obtained insively worse, and they finally die of massive cardiac necrosis.' " experimental cardiac failure where uncoupling of oxidativeOxidative phosphorylation (P /0 ratio) in the heart sarco- phosphorylation as well as a decrease in the high energysomes was determined, using a-ketoglutarate, malate and phosphate content have been demonstrated.'"pyruvate as substrates.' A significant depression in the P /0ratio of the myopathic hamster heart sarcosomes was obtained. 1. ~~d.b~hg::;;':C;;I.BE~~' (~as~i). ~:x339. C. W. and Whitney, R. (1962):The uncoupling of oxidative phosphorylation was substan- 2. Homburger, F., Baker, J. R., Nixon, C. W., Wilgram, G. andtiated by the findings that the adenosine triphosphate and Harrop, J. (1965): J. Path. Bact., 89, 133.creatine phosphate contents of these hearts were depressed, 3. Maley, G. F. and Lardy, H. A. (1953): J. BioI. Chem., 204, 435.4. Feinstein, M. B. (1962): Circulat. Res .. 10, 333.while the inorganic phosphate content was elevated. Further 5. Sehwartz, A. and Lee, K. S. (1962): Ibid., 10, 321.THE EFFECT OF ACUTE ETHANOL INTOXICATION ON THE METABOLISM OF PYRUVATE ANDPALMITATE IN THE PERFUSED RAT HEARTR. COWLEY, M.B., CH.B. (CAPE TOWN), F.c.P. (S.A.), M.MED. (lNT.) (STELL.), CSIR Degenerative Diseases Group, Universityot Stellenbosch Medical School, Karl Bremer HospitalIn order to determine if any biochemical abnormality exists in was converted to .C.140,.' The effect of lOOmM ethanol on bothalcoholic cardiomyopathy,"- the effect of alcohol on su.bstrate substrates was mlDlmal.metabolism in the isolated perfused .rat heart was studied. It is suggested that the high level of ethanol may affect theThe effect of different concentrations of ethanol (IOOmM, permeability of the cell membrane or the osmolarity of the200mM) on the metabolism of pyruvate-C' and palmitate-I-Cl< perfusion medium.'was studied, using a closed recirculation system.' It could be 1. Regan, T. J., Koroxenidis, G., Moschos, C. B., Oldewurtel, H. A.,demonstrated that the higher concentration of ethanol in the Lehan, P. H. and Hellems, H. K. (1966): J. Clin. Invest., 45, 270.perfusate depressed the metabolism of pyruvate, while having 2. Lleber, C. S. (1966): Gastroenterology, SO, 119.ff h k f I't t Th' t' f 3. Waklm, K. G. (1946). Fed. Proc., 5, 109.no e eet on t e upta e 0 pa ml a e. e IDcorpora .lOn 0 4. Ferrans, V. J., Hibbs, R. G., Weilbaecher. D. G., Black, W. C. andpalmitate into tissue lipid, however, was enhanced, while less Burch, G. E. (1965): Amer. Heart J., 69, 748.702 S.A. TYDSKRIF VIR GENEESKUNDE 6 Augustus 19663-Q-METHYL-~-RHAMNOSE AS A CONSTITUENT OF PLANT POLYSACCHARIDE GUMSM. KAPLAN, A. M. STEPHEN AND DAPHNE VOGT, Department of Chemistry, University of Cape Town+-1-++Trace+Trace++++b+Welwitschiamirabilis gumTrace++++++3, 4-tri-Q-methyIglucuronic+++++++++++++Traces+++( --+6Galp--+) --+3Galp--+") --+Ara(p or f)--+L--+GlupAc--+Sugar residueIRhamnopyranose--+End Arabinofuranose--+groups Galactopyranose--+lMannopyranose--+Glucopyranuronic acid--+( --+6Galp--+I 3I tII --+2Manp--+Branchl' 3points tI ij--+6G3alp--+L ta-In addition. an acidic component resembling 2.a:id appeared on the g.1. chromatogram.b--Several components involved.tentative assignments of some of the inter-sugar linkages in I andIII are given in a first attempt to compare the main structuralfeatures of the two polysaccharides:Encephalartoslongifolius gum"+++++TraceChainunitsWe acknowledge the collaboration of the Curator and Staffof the Bolus Herbarium, and are grateful to the CSIR for anassistantship.1. Reichstein. T. and Weiss, E. (1962): Adv. Carbo. Chem.. 17, 65.2. Aspinall, G. 0., Johnston, M. J. and Stephen, A. M. (1960): J. Chem.Soc., 4918.3. Shaw, O. H. and Stephen, A. M. (1965): S. Afr. Industrial Chemist,146.4. Jones, J. K. N. and Gorrod, A. R. N. (1954): J. Chem. Soc., 2522.5. Stephen, A. M., Kaplan, M .• Taylor, G. L. and Leisegang, E. C.(1966): Tetrahedron (in the press).6. Kaplan, M. (1965): M.Se. thesis, University of Cape Town.3-Q-Methyl-L-rhamnose (acofriose) is a component' of cardiacg1ycosides, bacterial glycolipids, and the hemicelluloses of Blackspruce (Picea nigra) wood. We report finding this sugar as a minorcomponent of a polysaccharide (I) found abundantly in radialcanals, and also as an exudate from the stems, of Encephalartoslongifolius Lehrn. (female), a member of the botanically significantfamily Cycadaceae. 3-Q-Methyl-!,..rhamnosewas shown as identicalto a specimen from methylatedKhaya senegalensis gum' by m.p.and mixed m.p., [ex1Q., paper- and thin-layer chromatography, i.r.spectrum and X-ray diffractometry. The derived glycitol gaveCH.O on 104- oxidation, and the methyl glycosides of the sugar,like those of the authentic specimen, were only partly unaffected by104- during 6 days (checked by g.l.c. against an internal standard).De-Q-methylation of the sugar gave rhamnose.Paper chromatography indicated' that 3-Q-methylrhamnose isalso present in the gum exudates, (JI) and (Ill) respectively, ofEncephalartos latefrons Lehm. (female) and of Welwitschia mirabilisHook f. (fam. Gnetaceae). It is known that the parent sugarL-rhamnose may be formed from Q-glucose (as thymidine diphos-phate derivative) without rearrangement of its carbon chain, andthe methyl ether may arise similarly from 3-Q-methylglucose(detected in a glycoside of Streblus asper).'E. longifolius gum (I) forms a voluminous, fragile gel on solutionin water, has [ct1!5-28°, and is an acid of equivalent weight 600.From an acid hydrolysate were isolated, in addition to 3-Q-methyl-!,-rhamnose, !,-rhamnose, I)-galactose, l-arabinose and I)-xylose(characterized as crystalline derivatives>;" while there is evidence(paper chromatographic) for Q-mannose, L-fucose, and uronicacid (probably I)-glucuronic,-linked f31-.:6 to galactose andf31--+2 to mannose). Hydrolysates of Il and III (Ill had [ex]0-45°and equivalent weight 1,5(0) showed a similar complexity of sugarcomponents; they resemble also the hydrolysate of Black sprucewood.'Portions of methylated I ([ex16-23° in CHCI3 ; OMe, 34·6%)and of methylated III ([ex1B-58°; OMe, 38·6%; prepared by Dr.D. H. Shaw) were (i) hydrolysed to give a number of methylatedsugar components which were separated and recognized by paperchromatography, and (ii) methanolysed and analysed semi-quanti-tatively' by gas liquid chromatography. The exceedingly complexchromatograms defy complete interpretation, but the followingEXPERIMENTAL PULMONARY HAEMOSIDEROSIS AND PULMONARY OEDEMAA. R. BOSMAN AND M. S. DUNNILL, The Departmenr of Internal Medicine, University of Ste/lenbosch and the Departmentof the NufJield Professor of Surgery and Department of Pathology, University of OxfordIn 1951 Magarey showed that when blood was given erido-tracheally to rats it was first distributed diffusely throughoutthe lung. Later focal aggregates of haemosiderin-Iaden macro-phages were formed, similar to those found in mitral stenosisand other conditions giving rise to chronic passive venouscongestion in the human lung. These findings confirmed thatfocal aggregates of siderophages could follow a diffuse hae-morrhage and were not necessarily the result of focal haemor-rhage from bronchioles as has been suggested by Lendrum,Scott and Park (1950) in patients with mitral stenosis. Mostauthors agree that in mitral stenosis the walls of the alveolisurrounding focal haemosiderin deposits show some interstitialfibrosis, and it has been shown that the elastica of thepulmonary vessels and the alveolar walls may show iron en-crustation. The absence of interstitial fibrosis and otherchanges reminiscent of the haemosiderosis of mitral stenosisin Magarey's rat lung experiments is not clear; however, ithas been suggested that it may be the result of a lack ofchronic venous congestion in the lung or due to the relativelyshort duration of the experiment.Experiments will be described where the thesis that pul-monary oedema may alter the reaction of the lung to haemo-siderin was examined.Three groups of male albino Wistar rats were used and theprocedures relating to each group were repeated twice weeklyfor a period of 18 weeks. After a further period of 4 weeksall the animals were killed with ether and the lungs wereexamined.Pulmonary oedema was produced in group I by means ofthe intraperitoneal injection of 0·4 ml./IOO mg. body-weightof 6% ammonium chloride. In the ether-anaesthetized animalsof group n, 0·75 m\. of heparinized rat blood was injectedendotracheally. In group ill the injection of 0·75 m\. of bloodendotracheally was followed immediately by the intraperito-neal injection of anImonium chloride in the same dose asfor group I. .A total of 70 rats were used for these experiments. Tenanimals in each group were allowed to complete the fullcourse, and 14 in each group were killed or died at intervalsduring the course of the study. Paraffin sections were cut at5 )l and stained with haematoxylin and eosin, Perls' method foriron and the Gomori method for reticulin.It was found that in animals given ammonium chloride-induced pulmonary oedema and endotracheal blood, the focalaggregates of siderophages took longer to form. Iron encrusta-tion of the connective tissue fibres occurred in these animals.No interstitial fibrosis was observed in the alveolar walls, butthe alveolar epithelium had a tendency to be cuboidal whereit surrounded a collection of siderophages. The findings inthese lungs will be compared with those in human cardiacpulmonary haemosiderosis.

Experimental biology group: Summaries of Scientific papers

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Experimental biology group: Summaries of Scientific papers

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