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A simplified procedure for filter paper electrophoresis is described in which disturbing factors such as evaporation, heating, buffer concentration gradients, and pH changes in the electrode vessels were reduced to a minimum. Artificial mixtures of highly purified proteins could be separated and the components isolated. The application of the method to a variety of studies on serum proteins is demonstrated. Protein concentration in paper segments was determined by two different methods of protein estimation. Curves were obtained showing the same five major peaks for normal serum as found by the classical methods of free electrophoresis. Comparisons were made of the areas of the various components under the curves obtained with the different methods. Two dimensional electrophoresis was applied to serum and serum components. It proved of value in demonstrating the heterogeneity of fractions such as the γ-globulin of serum. The polysaccharide dextran was used as an index of the extent of electro-osmotic flow during the course of the various experiments. The ratio of the distance of electroosmotic flow and the distance of protein migration was shown to be constant for a given type of paper. For serum albumin on Munktell 20 paper this ratio was 0.35. A formula for mobilities applicable to liquid in a highly porous supporting medium is presented. Mobility values for human serum albumin at various pH levels on paper showed approximate agreement with those obtained in free solution giving a similar isoelectric point.

The growth inhibition test for identifying Mycoplasma species has been modified by drying antibody-impregnated paper discs at 5 C. When stored at −20 C, these discs have been found to retain their inhibitory activity for longer than 7 months. Since these discs can be stored for long period of time, significant advantages over present methods result. When, for example, discs are arranged on a ring, a single test can be used for the identification of an unknown human species. Valuable antisera can be distributed to other laboratories on paper discs in much less volume than can fluid antiserum. Considerable savings of time result from prior preparation of many discs that can then be stored and used over a long period of time. The growth-inhibiting antibody is stable, and the activity is not enhanced by a heat-labile accessory factor from fresh guinea pig serum which increases the antibody titer in the metabolic inhibition test.

Microbiological assay of individual antibiotics in mixtures of antibiotics depends on the use of selective inactivation and/or of test bacteria with differential susceptibility. Controlled experiments revealed that streptomycin in concentrations of 20 and 40 μg/ml did not influence a disk diffusion assay of penicillin with Sarcina lutea (ATCC 9341) as the test organism. In the case of penicillin concentrations less than or equal to 1 IU/ml, addition of 80 μg of streptomycin per ml influenced the penicillin assay significantly. Clinical use of streptomycin resulting in levels above 40 μg/ml usually did not occur; therefore penicillin could be assayed as though streptomycin were not present. We observed additionally that S. lutea was unable to grow on agar plates prepared with semicarbazide hydrochloride.

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Experiências foram realizadas com bactérias dos gêneros Listeria e Erysipelothrix, em meios líquido e sólido, utilizando o clorêto de 2, 3, 5 - trifeniltetrazólio. A atividade enzimática redutora das listérias para o TTC foi diferente da do E. rhusiopathiae, principalmente em meio líquido e nas horas iniciais de observação. Preparações feitas para microscopia ótica e electrônica dos germes tratados com TTC revelaram a presença de granulações polares, bipolares e centrais dentro do corpo das listérias. A evidenciação de granulações coradas de formazana, intracelulares nas listérias, confirma estudos anteriores quanto à possibilidade da existência de mitocôndrias nas bactérias.Based on a similarity of cultural and biochemical properties, WILSON and MILLES (6) compared the microorganisms of listeriosis with those of swine erysipelas including them in the genus Erysipelothrix with the species E. monocytogenes and E. rhusiopathiae. However, BARBER (1) and JULIANELLE (4), in a comparative study of both microorganisms, do not admit that possibility. Observing the morphological and citochemical characteristics of L. monocytogenes (N.º 7 973, 5 348, 5 105, 5 214, of the Seeliger collection) and E. ehusiopathiae cultures (N.º 1, 7, 11, 27, 37, of the WIX collection), both in phase "S", we were able to verify their comparative behavior when in contact with 2-3-5-triphenyltetrazolium chloride (TTC), "Synthetical Laboratories, Chicago, U.S.A". 1. To 1,0 ml of cultures of both microorganisms grown in plain broth plus 5% horse serum for 24 hours at 37ºC, 0.1 ml of sterilized 1% aqueous solution of TTC was added. The tubes were left at room temperature (± 25ºC) or in the incubator (37ºC). In the tubes with L. monocytogenes immediately appeared a red coloration (fig. 1) characteristic of formazan production, that became gradually more pronounced, from bottom to top (figs. 2, 3, 4, 5); at the and of 24 hours the color was intense the tube. In the tubes with E. rhusiopathiae (figs. 1, 2, 3, 4), only a faint pink coloration was seen after 2 hours; at the end of 24 hours, the intensity of color was not equal to that observed in tubes with L. monocytogenes. 2. Petri dishes with 15 ml of plain agar with 5% horse serum, containing 0.1 ml of 1% sterilized aqueous solution of TTC, were inoculated with L. monocytogenes and E. rhusiopathiae, and incubated for 24 hours at 37ºC. Colonies of L. monocytogenes that developed presented a red color, indicative of formazan, as previously described by GRAY et al. (3), while the E. rhusiopathiae colonies did not show any coloration. 3. Disks of filter paper, previously soaked with TTC and dried were placed over the growth of L. monocytogenes in Petri dishes with plain agar plus 5% horse serum form 24 hours. No change in the color of the colonies was observed. The same negative result was obtained when the porous clay cylinder technique for measuring antibiotic activity was employed, With E. rhusiopathiae also negative results were obtained with the disk technique. 4. Wet preparations of the cultures after 1 hour of contact with TTC in the same conditions of item 1, were observed at 900X magnification. L. monocytogenes presented normal shape and dimensions, but one to three intracellular polar, bipolar and or central, red-purple granules were noted. E. rhusiopathiae bacilli were morphologically normal…

In vivo metabolic studies on rats and mice revealed a marked difference in the fluorescent compounds produced after ingestion of aflatoxin B(1). The mouse converted aflatoxin B(1) to three unknown fluorescent compounds, designated x(1), x(2) and x(3) and the known aflatoxin M(1), while the rat was only capable of producing aflatoxin M(1). The results suggested that metabolites x(1), x(2), x(3) and aflatoxin M(1) were not part of a major metabolic pathway, but produced independently. These unknown yellowish-green fluorescent compounds did not seem to be conjugated with sulphate or glucuronic acid.In vitro incubations of various mouse liver cell fractions with aflatoxin B(1) showed that metabolites x(1), x(2), x(3) and aflatoxin M(1), could only be produced by the microsomal fraction and that NADPH was needed as a co-factor. The differences in aflatoxin metabolism by mice and rats are discussed in relation to the apparent resistance of the mouse to the carcinogenic effects of this toxin.

1. A method involving the use of triple-labelled derivatives has been developed for the determination of total oestrone and oestradiol in the plasma of the domestic fowl. The double-labelling technique devised by Svendsen (1960) for the determination of free oestrogens in human plasma was modified to enable the total oestrogen recovery to be determined for each sample. 2. [6,7-(3)H(2)]Oestradiol-17beta is added to the plasma samples (1-10ml.), which are hydrolysed with acid and the phenolic steroids then extracted and partially purified. The extract is esterified with iodobenzene-p[(35)S]-sulphonyl chloride of high specific activity. After addition of standard oestrogen [(131)I]iodobenzene-p-sulphonates the esters are finally purified by paper chromatography. 3. The oestrogens are determined by comparing the (3)H/(35)S and (131)I/(35)S ratios in the purified esters with similar ratios of appropriate standards. 4. With this procedure the recoveries of oestrone and oestradiol after hydrolysis were 70-85% and 72-84% respectively, and after hydrolysis and preliminary purification 38-53% and 39-51% respectively. With this procedure up to 500ng. of oestradiol can be determined. The sensitivity of the technique for oestrone is 3.0ng. and for oestradiol 2.1ng. 5. The ranges of oestradiol and oestrone concentrations found in six plasma samples were 8.3-21.4ng./ml. and 15.2-31.6ng./ml. respectively.

Weinman, Donald E. (University of Georgia, Athens), George K. Morris, and William L. Williams . Unidentified growth factor for a lactic acid bacterium. J. Bacteriol. 87: 263–269. 1964.— Lactobacillus bulgaricus Georgia strain required an unidentified growth factor, named Georgia bulgaricus factor (GBF), when grown on a semisynthetic basal medium. Aqueous extracts of torula yeast and beef liver were the best sources of GBF. Adenine, ribonucleic acid (RNA), alkaline digests of RNA, and Mg ++ also stimulated growth, but to a considerably lesser extent than liver and yeast extracts. Several purines, pyrimidines, and related compounds also promoted growth responses in individual experiments, but not consistently. Intact deoxynucleic acid (DNA), deoxyadenylic acid (dAMP), and deoxyguanylic acid (dGMP) inhibited growth. The DNA and dAMP inhibitions were fully reversed by crude sources of GBF, while dGMP inhibition was only partially reversed. RNA reversed DNA inhibition to a small extent. GBF was stable to heat at pH 2.5 to 11 and to prolonged light exposure. It was destroyed by heating at pH 1.0. The GBF activity moved as a single component in paper chromatography. It was firmly adsorbed on charcoal and poorly soluble in organic solvents. A concentrate, 28 times more potent than liver extract, was prepared by prolonged paper chromatography. All known growth factors and biological compounds readily available were assayed for GBF activity, none of which gave a response similar to the crude extracts. Final proof that a new growth factor exists must await definite identification of the active compounds.

Various observations cited at the beginning of this paper and in the Discussion suggested that phalloidine, a poisonous constituent of the green mushroom Amanita phalloides, would not be toxic by itself, but would be rendered toxic previously in the liver. Using a tritiated toxic derivative of phalloidine ([3H]desmethylphalloin) we found that: (a) in rats, 60% of the administered substance are excreted unchanged in the urine within the first day. A second radioactive substance of low concentration, moving more slowly in paper chromatography, turned out as a product of the self-degradation of [3H]desmethylphalloin; (b) respectively 98% and 99,9% of the radioactivity were extracted with methanol from homogenates of livers of rats or mice which had received [3H]desmethylphalloin 1 to 2 hours before. Aqueous homogenates of liver of poisened rats also contained radioactivity firmly bound to a high molecular weight substance, as shown by gel chromatography. The radioactive substance was totally removed from the carrier, presumably ribosomal material, by adding methanol, and was identified as [3H]desmethylphalloin; (c) [3H]desmethylphalloin was not metabolized on incubation with oxygen and rat liver ribosomes in a NADPH-regenerating system. A radioactive substance extracted from liver microsomes of a poisoned rat also proved to be original toxin. As a result of these experiments it can be concluded that [3H]desmethylphalloin and most probably also phalloidine are not metabolized in the livers of rats and mice.

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[14C]tyrosine and L-[14C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [14C]dopamine, [14C]norepinephrine, [14C]epinephrine, [14C]serotonin, [14C]tyramine, and [14C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.

When monolayers of freshly obtained rabbit lung macrophages are exposed to the nucleoside analogue, showdomycin (sho), adenosine transport, measured over a 45 s interval, is irreversibly inhibited. Low doses of the drug or short periods of exposure, however, do not result in decreased transport, while higher concentrations or longer exposures result in exponential decline. The initial lag is not due to a long reaction time of sho with the transport carrier or to nonspecific sites absorbing the drug. Previously it was shown that preincubation of monolayers with normal rabbit serum (NRS) results in increased adenosine transport. When monolayers are first exposed to sho so as to inhibit transport to varying degrees and then incubated with NRS, transport is increased over the inhibited level. Several experiments make it unlikely that serum removes the drug from the cell surface in a nonspecific fashion. Moreover, serum given before, during, or after sho alters the dose response curve so that no shoulder is seen. One way to explain these results makes use of target theory: the adenosine transport system could be comprised mainly of "coupled" or "clustered" sites of which only one is active at any time as well as "hidden" sites which are inactive. When a site in a group is irreversibly inactivated by sho, another in the group becomes activated. Serum might activate or uncouple all sites and also cause the appearance of hidden sites, which previously neither transported nor bound sho.

Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11ß-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11ß-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact.

Goldfish retinas incubated with L-glutamate-14C (UL) were found to synthesize γ-aminobutyric acid-14C (GABA-14C) The accumulation of newly synthesized GABA was enhanced by physiological stimulation of the retina with flashing light; and this increase was directly proportional to the logarithm of the light intensity. The total GABA content was also higher in light-stimulated than in dark-adapted retinas, although the glutamate content remained unchanged No differences were found in the cell-free activities of glutamate decarboxylase (EC 4 1.1 15) and GABA-glutamate transaminase (EC 2.6.1.19) extracted from light-stimulated and dark-adapted retinas. These findings, together with other physiological and morphologcal evidence, suggest that GABA plays a functional role in synaptic transmission in the goldfish retina

Uptake of 35 S-hydroxyethylthiazole (4-methyl-5-hydroxyethylthiazole) by Escherichia coli intact cells was studied. Hydroxyethylthiazole was taken up in the presence and absence of glucose at the same rate. The uptake was almost proportional to a hydroxyethylthiazole concentration gradient up to 0.1 mM with no tendency of saturation, and reached a steady state within 2 min. When the cells were treated with 1 mM N -ethylmaleimide, about 50% inhibition of hydroxyethylthiazole uptake was observed. Hydroxyethylthiazole uptake was stimulated by the addition of hydroxymethylpyrimidine (2-methyl-4-amino-5-hydroxymethylpyrimidine), and this effect was further enhanced in the presence of glucose. For full activation of hydroxyethylthiazole uptake, 1 μM hydroxymethylpyrimidine was necessary in the presence of glucose. The rate of hydroxyethylthiazole uptake was almost linear up to 60 min in the presence of hydroxymethylpyrimidine and glucose. Hydroxymethylpyrimidine monophosphate and its pyrophosphate could not stimulate the uptake. Thiamine and 2-amino-hydroxyethylthiazole were inhibitory on hydroxyethylthiazole uptake in the presence of hydroxymethylpyrimidine and glucose. N -ethylmaleimide and 2, 4-dinitrophenol were also inhibitory. No stimulatory effect of hydroxymethylpyrimidine on hydroxyethylthiazole uptake was observed in mutant cells lacking either thiaminephosphate pyrophosphorylase or hydroxymethylpyrimidine monophosphate kinase. The possibility of direct participation of thiamine-synthesizing enzymes in hydroxyethylthiazole uptake was discussed.

In attempting to evidence the evolution and the structure--function relationships of the ribosome in procaryotes the authors have undertaken a comparative amino acid sequence analysis of ribosomal proteins from Escherichia coli, and Bacillus stearothermophilus and Halobacterium cutirubrum, organisms which differ substantially in their physiological tolerances and taxonomic relationships. Previous structural studies have indicated a high degree of homology in some of the 30 S ribosomal proteins from E. coli and B. stearothermophilus. Reported in this paper is a summary of results on the study of the amino terminal regions of 19 ribosomal proteins E. coli strain Q13 and 21 from B. stearothermophilus strain 10.

It is well known that the change in external pressure is one of the major factors in diving accidents. The mechanisms by which this creates such conditions as depth narcosis, rupture of the lung, and decompression sickness are either known or under investigation. Nevertheless, many accidents cannot be explained in terms of classical diving hazards. This paper concerns a reaction to pressure change that has hitherto been largely overlooked as a potential cause of such accidents. Although reports were published as early as 1896 by Alt and in 1909 by Keays of vertigo occurring at the end of exposure to increased pressure environment, there are few accounts of this subject in the literature on labyrinthine func tion or on diving medicine. Fields (1958) reports four cases of brief vertigo in connexion with surfacing after dives, and Coles and Knight (1961) recorded three cases in connexion with Navy divers' clearing their ears. Rowe (1961) also men tions that vertigo may occur in divers, and reports one case without discussing the specific cause.

1. Incubation conditions were established in experiments with human-prostate homogenates for almost complete conversion of [4-(14)C]testosterone into at least ten transformation products. 2. Whole homogenates of tissue with benign hypertrophy were shown to contain 3alpha-, 3beta- and 17beta-hydroxy steroid dehydrogenases, Delta(4)-3-oxo steroid 5alpha- and 5beta- reductases and unidentified hydroxylases. 3. Most of the 17beta-hydroxy steroid-dehydrogenase activity was located in the mitochondria, which showed little other activity. 4. The 3alpha- and 3beta-hydroxy steroid dehydrogenases and the 5beta-reductase were located in the high-speed supernatant and required supplementation with NADPH for activity. 5. The 5alpha-reductase was located in both microsomal and high-speed supernatant fractions and also required supplementation with NADPH.

The origin of desacetylcephalosporin C in cephalosporin C fermentation broths was investigated. Esterase activity was detected in cell-free extracts of Cephalosporium acremonium , but these extracts failed to deesterify cephalosporin C. When cephalosporin C was added to sterile and inoculated fermentation media, the antibiotic decayed at nearly identical rates. The formation of desacetylcephalosporin C during the fermentation was measured by quantitative chromatography and by the incorporation of valine- 1 - 14 C into the molecule. The rate constants obtained from the results of these experiments were equivalent to those for the decay of cephalosporin C in sterile and inoculated media. The data demonstrate that desacetylcephalosporin C is produced by nonenzymatic hydrolysis of cephalosporin C.

The tRNA methylase activity in vitro of adenovirus-18-induced tumour was studied. The activity expressed per mg of protein was the same for extracts of tumours induced by either adenovirus-12 or adenovirus-18. The methylated bases isolated after incubation with extract from adenovirus-18-induced tumour were N1-methylguanine, 1-methyladenine, 3-methyladenine and N6-methyladenine.

Preparations of membrane lipoteichoic acid containing different amounts of protein were isolated from intact organisms of Lactobacillus fermenti NCTC 6991 by various procedures chosen for their ability to disrupt the hydrophobic interaction of lipoteichoic acid with other membrane components. The highest yield of lipoteichoic acid was obtained with hot aqueous phenol, and this preparation contained very little protein. Partial removal of cell lipids with chloroform-methanol followed by extraction with water at 100 C gave a lipoteichoic acid-protein complex that was a very effective immunogen; immunogenicity was shown to relate to protein content, though the specificity of the antibodies was directed against the teichoic acid component.

After infection of Escherichia coli with T4 phage, phospholipid synthesis continued but at a reduced rate. The same phospholipid components were synthesized as in uninfected cells; however, the relative rates of 32 P i incorporation into phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were altered. This alteration was most pronounced during the first 10 min after infection. Under these conditions, the isotope incorporated into PG equaled or exceeded that found in PG from uninfected cells. Chloramphenicol (CM) added before, but not 5 min after, infection inhibited the relative increase in PG synthesis, and CM added at different times after infection indicated that a protein synthesized between 3 and 6 min was required for this change to occur. Supplies of exogenous l -serine or l -α-glycerol-P failed to affect the relative rates of 32 P i incorporation into PG and PE by infected or uninfected cells. Phospholipid synthesis was somewhat higher after infection with T4rII mutants than after infection with wild-type phage. After infection with these mutants or several amber mutants, the relative synthesis of PG and PE was characteristic of T4r + -infected cells. The phospholipid synthesized after infection did not rapidly turn over, but infection accelerated the loss of PG synthesized prior to infection.

Three pteridines have been isolated from the methane- or methanol-oxidizing bacterium Methylococcus capsulatus. Two of these are known compounds, 2-amino-6-carboxy-4-hydroxypteridine and 2-amino-4-hydroxy-6-methylpteridine. The third is shown by degradative and synthetic experiments to be l-threo-neopterin 2′:3′-phosphate. Labelling experiments show that both the pteridine moiety and phosphate residue are derived from a single GTP molecule. The possible metabolic significance of these compounds in methanol oxidation is discussed.

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin–Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3–9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a `cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.

The kinetic properties of acyl-coenzyme A (CoA): l -α-glycerol-phosphate trans-acylase (EC 2.3.1.15) from Escherichia coli were studied. At 10 C, a temperature at which the reaction was proportional to time and enzyme concentration, the enzyme had an apparent K m of 60 μ m for l -α-glycerol-phosphate. The curve describing the velocity of the reaction as a function of palmitoyl-CoA concentration was sigmoid but the plot of v −1 versus [S] −3 gave a straight line. A K m of about 11 μ m was calculated for palmitoyl-CoA. Adenosine triphosphate specifically inhibited the reaction, being a noncompetitive inhibitor in respect to l -α-glycerol phosphate. Inhibition only occurred with high concentrations of palmitoyl-CoA, and maximal inhibition was 60%.

Three aggregational forms of arylamidase are produced by Cephalosporium acremonium . The exocellular enzyme, with an approximate molecular weight of 60,000, was purified 300-fold by diethylaminoethyl cellulose chromatography, gel filtration, and gel electrophoresis. With l -leucyl-β-naphthylamide as the substrate, the K m is 4.2 × 10 −4 m ; the optimum p H, 7.7; and the temperature optimum, 35 C. The enzymatic hydrolysis of l -leucyl-β-naphthylamide is inhibited by a number of cephalosporins, whereas a variety of penicillins show no effect. Alternatively, the enzyme specifically catalyzes the β-lactam hydrolysis of a number of cephalosporins; a number of penicillins are resistant. The K m for cephalosporin C is 9.09 × 10 −4 m .

In 1929, Fleming discovered penicillins and observed that this group of substances kills gram-positive bacteria more effectively than gram-negative bacteria (thus implying some important physiological difference between the two groups of organisms). I Subsequent knowledge of the bacterial cell wall made it possible to establish both on physiological and chemical grounds that penicillins are specific and highly selective inhibitors of the biosynthesis of cell walls, both in gram-positive and in gram-negative bacteria.2-6 The reason for the relative insensitivity of gramnegative bacteria to most penicillins has remained obscure. More recent knowledge of the structure and biosynthesis of bacterial cell walls has suggested that, in a terminal reaction in cell wall synthesis, linear glycopeptide strands are cross-linked in a transpeptidation, accompanied by release of the terminal D-alanine of the pentapeptide precursor, with formationi of a two- or threedimensional network. Direct chemical analyses of cell walls prepared from cells treated with penicillinll 8 and isotopic studies of wall biosynthesis9' 10 suggested that penicillin was interfering with this hypothetical g]ycopeptide cross-linking reaction. In the presence of penicillin, nascent glycopeptide, an uncross-linked mlonomeric uniit of the wall, accumulated.8 Pulse-labeling experiments have established that this nascent unit is an immediate precursor of the final cross-linked glycopeptide; penicillin completely inhibited its integration into the iietwork.10 Moreover, molecular models of penicillin resembled the acyl-D-alanyl-D-alanine in the ]inear glycopeptide and it was possible to suggest a molecular mlechanism for the transpeptidation and its inhibition by penicillin.8 However, the actual reaction or reactions inhibited had not been demoonstrated. In this paper we wish to report that a cell-free enzymatic system has been obtained from ce]ls of Escherichia coli which catalyzes this cross-linking reactiorn. We call the enzyme which catalyzes this step glycopeptide transpeptidase, since it catalyzes a reaction in which the peptide chains of two linear glycopeptide strands are cross-linked by a transpeptidation, in which the terminal D-alanine residue of one of the strands is elimirnated; this peptide bond synthesis proceeds without any other source of energy and is reversible. The terminal D-alanine residue of the other strand is also removed by hydrolysis catalyzed by a D-alanine carboxypeptidase in the preparation (Fig. 1). Both of these reactions are inhibited by low levels of penici]lin G, other penicillins, and a cephalosporin. Materials and Methods.-Preparation of substrates: UDP-MtirNAc L-ala -D-glu H3-mesoDAP D-ala D-ala and UDP-MurNAc L-ala- D-glu meso-DAP C14-D-ala C14-D-ala were prepared enzymatically by sequential addition of amino acids to the appropriate uridine nucleotides. lOa UJDP-GlcNAc-C14 is the same preparation uised previously. Preparation of enzyme: Two particulate enzymes, obtained from cells of E. coli strain Y-1O or E. coli strain B, were employed. The cells were ground with alumina. The fraction of the dis

In certain strains of Staphylococcus aureus , a concentration of erythromycin between 10 -8 and 10 -7 M can induce resistance to concentrations of this drug as high as 10 -4 M. In one such strain studied, S. aureus (1206), N 6 -dimethyladenine is not normally present in 23S rRNA; however, a compound presumptively identified (on the basis of paper chromatography in three different solvents) as N 6 -dimethyladenine appears in the 23S rRNA of growing cells that have been incubated in a medium containing 10 -7 M erythromycin. It has been shown previously that the induction of the erythromycin-resistant phenotype that occurs under these conditions requires 10 -8 -10 -7 M erythromycin for maximal expression within 1 hr and that induction results in modified 50S ribosomal subunits, which are then unable to bind erythromycin or lincomycin. Methylated adenine is also found in the 16S rRNA from the strain of S. aureus studied; however, in contrast to the situation with 23S rRNA, the amount in 16S rRNA is not affected by erythromycin. These findings provide the first example of a correlation between the methylation of rRNA and altered ribosomal function.

Neurospora produces a mucopolysaccharide (called MP) which inhibits its growth, causes vacuolation and agglutination of its cells, and precipitates its purified membrane protein. Cultures of a colonial strain display a phase of slow growth; the induction of this phase is traced to the production of MP by the mold. Stationary-phase cultures of wild type also produce MP. MP is a polymer of galactosamine, its amino groups only partially acetylated, probably containing other minor components. MP molecular weight is approximately 10 6 . Complete acetylation abolishes the biological activities of MP. It is suggested that the regulatory effect of MP is mediated by its interaction with the protoplasmic membrane.

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two p H optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.

A threonine dehydrataseless mutant of Escherichia coli , Crookes strain, was observed to grow on an acetate minimal medium without the usual requirement for isoleucine supplementation. Both the wild-type Crookes strain and a threonine auxotroph metabolized l -glutamate- 1 - 14 C to l -isoleucine- 1 - 14 C with no appreciable randomization, suggesting that a pathway for isoleucine formation from glutamate via β-methylaspartate, β-methyloxaloacetate, and α-ketobutyrate was possible in addition to the pathway from threonine and α-ketobutyrate. Crude cell-free extracts formed 14 C-β-methylaspartate from 14 C-glutamate, and the conversion of β-methylaspartate to α-ketobutyrate was also demonstrated, thus supporting the conclusion that glutamate can serve as a precursor of α-ketobutyrate (and isoleucine) without the necessary involvement of threonine as an intermediate.

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.

The complete amino acid sequence of wool protein SCMKB-IIIB3 was determined. The peptides used for the sequence work were obtained by peptic and thermolysin digestions and were fractionated by chromatography on DEAE-cellulose, paper chromatography and electrophoresis. The peptides were analysed by dansyl–Edman degradation, mass spectrometry and tritium-labelling of C-terminal residues. The protein consists of 98 residues and has acetylalanine as N-terminal residue and carboxymethylcysteine as C-terminus. It is homologous with protein SCMKB-IIIB2 (Haylett & Swart, 1969). A salient feature of the sequence of protein SCMKB-IIIB3 is three consecutive cysteine residues.

Seven patients with hemoglobin S-thalassemia disease belonging to three Greek families are described. Interaction of the genes for hemoglobin S and for thalassemia was evident in all but one cases. Hemoglobin analyses consisting of a combination of paper electrophoresis and the alkali denaturation technic revealed the S+A+F pattern in three, the S+F+A pattern in one, the A+S+F in one, the S+A in one and the S+F pattern in another case. The importance of the hemoglobin pattern for the differential diagnosis of sickle cell-thalassemia disease from the sickle cell trait and the homozygous sickle cell anemia is discussed. From the genetical point of view the existence of modifying thalassemia genes is discussed. © 1965 J. F. Lehmanns Verlag.

Cultured mouse peritoneal macrophages were found to be relatively impermeable to streptomycin. Based on radioactivity measurements and radioautographic evidence, macrophages were impermeable to tritiated dihydrostreptomycin for periods up to 20 hr of incubation. Little or no intracellular streptomycin could be detected even when incubation was carried out in the presence of therapeutic blood levels of carrier dihydrostreptomycin. When the cultured mouse macrophages were allowed to phagocytize staphylococci, yeast cells, or polystyrene latex particles in the presence of tritiated streptomycin, the impermeability of the cells to the antibiotic was not affected. These observations suggested that the process of phagocytosis does not facilitate the intracellular accumulation of streptomycin, as seems to be the case for the fixed phagocytic cells of the liver.

The prevalence of tetracycline-resistant beta-haemolytic streptococci in South-west Essex has been recorded over the past 10 years. It has fallen from a peak of 35% in 1965 to a level of 9·2% in 1972. Ear infections no longer provide the highest incidence of these organisms; vaginal, perineal, and skin infections now seem to be of greater relative importance but throat swabs still provide the greatest actual number of isolations. Erythromycin-resistant strains are still rare.

During an ultrastructural study of small-intestinal mucosa from a patient suffering from alpha-chain disease organisms were identified within the epithelial cytoplasm which showed the fine structural features of the coccidian group. Though coccidiosis is well recognized as causing a diarrhoeal and often lethal illness in animals it has been neglected as a cause of disease in man. Thus this finding may be significant and warrants further investigation into its possible role in the pathogenesis of alpha-chain disease.

The serum muramidase levels were measured in 128 patients with primary or metastatic colorectal cancer, 166 tumour-free patients after resection of a colorectal cancer, and 172 controls. Muramidase levels over 10 mug/ml were detected in 30%-39% of the tumour-bearing patients, in 8.2% of the tumour free, and in only 1.7% of the controls (normal level 6.68 +/- 1.42 mug/ml). Long-term follow up indicated that raised levels may occur as a transient phenomenon in recurrent or metastatic disease. The likely relation of abnormal serum muramidase activity and stimulation of the reticuloendothelial system is discussed.

Management of a patient with snake bite is influenced by the nature of the offending snake. Species diagnosis based on the patient's history and physical signs is often unreliable and the possibility of making a species diagnosis by immunological means has therefore been investigated. Wound aspirates, blister fluids, sera, and urine samples from patients with snake bite were examined for the presence of species-specific venoms using immunodiffusion. A positive species diagnosis was made in 40 out of 101 patients. Immunodiagnosis was especially successful in patients bitten by the puff adder, Bitis arietans, and the African spitting cobra, Naja nigricollis. A higher success rate could probably be achieved using more specific antisera and more sensitive assay techniques.

Micro-injection into the oocytes and eggs of Xenopus laevis was used to ascertain the effects of synthetic polyribonucleotides on protein synthesis in living cells. Poly(U) and poly(A) were not translated detectably, nor did they change the rate of endogenous protein synthesis. The same was true of poly(G,U), poly(A,G,U), poly(A,C,G,U), G-U-G-(U)n, A-(U)n and AUG. In contrast, A-U-G-(U)n was a potent inhibitor of protein synthesis in the cell. This might be because it is initiated normally but lacks a termination codon, or because it inhibits the translation of other molecules in some way not dependent on its normal initiation. Poly(G,U), poly(A,G,U) and poly(A,C,G,U) inhibited haemoglobin synthesis when they were injected into the oocyte with haemoglobin mRNA. The synthetic polyribonucleotides did not inhibit the translation of the natural mRNA when the two sorts of molecules were injected at different times. It is suggested that the synthetic RNA molecules compete with the natural mRNA for a pre-initiation factor in limited supply.

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy- d -glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K m of 7 μM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa . Various experimental data clearly indicated that P. aeruginosa PAO transported methyl α- d -glucose (α-MeGlc) via the glucose transport system. The apparent K m of α-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for α-MeGlc than for glucose. While only unchanged α-MeGlc was detected intracellularly in P. aeruginosa , α-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.

After the remission of symptoms and reduction in spleen size while on proguanil therapy four patients with the tropical splenomegaly syndrome defaulted from treatment. The withdrawal of proguanil caused a recrudescence of original symptoms, splenomegaly, and a return of the initially raised serum IgM. Complete return to normal values was again effected with proguanil therapy. The role of the spleen in the tropical splenomegaly syndrome in the production of the raised serum IgM is discussed. These patients should be educated as to the nature of their disease and the importance of continued medical treatment.

Cell-mediated immune responses to Tamm-Horsfall glycoprotein isolated from human urine were investigated using the leucocyte migration test. Abnormal responses were found in 91% of patients with active chronic hepatitis or primary biliary cirrhosis with an associated renal tubular acidosis (R.T.A.) but in only 19% of those without R.T.A. In nearly all of a group of patients without autoimmune liver disease and in a control group of normal subjects results were within normal limits. In addition, using an immunofluorescent technique with rabbit antibody to human Tamm-Horsfall glycoprotein, it was possible to show the presence in human liver cell membrane of material reacting immunologically as Tamm-Horsfall. These findings suggest that the development of an immune response to this glycoprotein, initiated by release of cross-reacting antigens from damaged hepatocytes, could be the mechanism underlying the occurrence of R.T.A. in some patients with autoimmune liver disease.