Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

Effects of isoniazid on the composition of mycobacteria, with particular reference to soluble carbohydrates and related substances

1. When growing Mycobacterium tuberculosis BCG was exposed to 0.5-10mug. of isoniazid/ml. there was intracellular accumulation of soluble carbohydrate, combined phosphate and substances absorbing at 260mmu. Yellow pigments were formed when modified Sauton medium was used, but not with Proskauer & Beck medium. These processes were apparent after 1hr. but were more marked after about 6hr. These effects were not found with an isoniazid-resistant strain. 2. After 6hr. exposure of the sensitive strain to 10mug./ml. there was little change in the amounts (per g. of insoluble nitrogen) of total lipid, glycolipid, RNA, DNA or of carbohydrate in the nucleic acid fractions. 3. The major accumulation was of alphaalpha'-trehalose. There was also accumulation of glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, trehalose 6-phosphate (tentatively identified), a polysaccharide containing only glucose, and an oligosaccharide containing glucose and glucose 6-phosphate, but not of glycerol and glycerol 3-phosphate. The u.v.-absorbing materials appeared to be nucleotide sugar derivatives. 4. In Mycobacterium smegmatis a similar accumulation of trehalose occurred on exposure to isoniazid, but there was little accumulation of other compounds. 5. No evidence could be found that isoniazid specifically affected the oxidation of glycerol or glycerol 3-phosphate. 6. It is suggested that the primary action of isoniazid on mycobacteria may be partial inhibition of a reaction in some central area of metabolism, such as glycolysis.     

In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.     

Regulation of two aspartokinases in Bacillus subtilis

When grown on minimal glucose medium, transformable Bacillus subtilis strains contained two distinct aspartokinases (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4). One of these enzymes was inhibited by l-lysine (Lys), whereas the other was insensitive to inhibition but was activated by l-leucine. None of the other amino acids tested had any effect, and the addition of l-threonine did not enhance the inhibition by Lys, in contrast to the concerted inhibition observed for other bacilli. At the end of exponential growth, the Lys-sensitive aspartokinase activity decreased, whereas the Lys-insensitive activity remained relatively constant throughout the stationary phase. The two activities were separated by (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Growth in the presence of Lys reduced the specific activity of aspartokinase by about 50% and eliminated the inhibition by Lys. In extracts of these cells, only Lys-insensitive activity was found upon (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Lys apparently repressed the synthesis of the Lys-sensitive enzyme.     

The purification of cholinesterase from horse serum

A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85-90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.     

Effect of Iron and Salt on Prodigiosin Synthesis in Serratia marcescens

Serratia marcescens wild-types ATCC 264 and Nima grew but did not synthesize prodigiosin in a glycerol-alanine medium containing 10 ng of Fe per ml. Wild-type 264 required the addition of 0.2 mug of Fe per ml for maximal growth and prodigiosin synthesis; Nima required 0.5 mug of Fe per ml. Three percent, but not 0.1%, sea salts inhibited prodigiosin synthesis in a complex medium containing up to 10 mug of Fe per ml. NaCl was the inhibitory sea salt component. The inhibition was not specific for NaCl; equimolar concentrations of Na(2)SO(4), KCl, and K(2)SO(4) also inhibited prodigiosin synthesis. Experiments with strains 264 and Nima and with mutant WF which cannot synthesize 4-methoxy-2-2'-bipyrrole-5-carboxyaldehyde (MBC), the bipyrrole moiety of prodigiosin, and with mutant 9-3-3 which cannot synthesize the monopyrrole moiety 2-methyl-3-amylpyrrole (MAP) showed that both MBC synthesis and the reaction condensing MAP and MBC to form prodigiosin were relatively more sensitive to NaCl inhibition than the MAP-synthesizing step. The capacity of whole cells to condense MAP and MBC was present, but inactive, in cells grown in NaCl; removal of the NaCl from non-proliferating salt-grown cells restored the activity. Other evidence suggests the existence of a common precursor to the MAP- and MBC-synthesizing pathways.     

Production of Types A and B Spores of Clostridium botulinum by the Biphasic Method: Effect on Spore Population, Radiation Resistance, and Toxigenicity

Spores of three strains each of type A and type B Clostridium botulinum were produced both by a biphasic (solid medium overlaid with an aqueous phase) and by a "conventional" (deep broth culture) procedure. Sporogenesis by the biphasic system was more rapid, convenient, and economical, and yielded as many or more heat-resistant (80 C, 10 min) spores per milliliter as by the conventional technique. Of several aqueous phases [thiamine-hydrochloride, yeast extract, (NH(4))(2)SO(4)] tested with strain 62A, the highest spore colony counts were obtained with 2.0% (NH(4))(2)SO(4). The six strains formed maximum spore numbers in 5 to 6 days of incubation. Spores produced by the two methods had essentially equal radiation resistances (D and lag values), and their subcultures gave similar toxin titers (LD(50) values).     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Enhanced antibiotic activity of Xenorhabdus nematophila by medium optimization

Nutrition had highly influence on the antibiotic production by Xenorhabdus nematophila YL001. Glucose and peptone were identified as the best carbon and nitrogen sources that significantly affected antibiotic production using one-factor-at-a-time approach. Response surface methodology was applied to optimize the medium constituents (Glucose, peptone and minerals) for antibiotic production by X. nematophila YL001. Higher antibiotic activity (328.9 U/ml) was obtained after optimizing medium components. The optimal levels of medium components were (g/l): glucose 6.13, peptone 21.29, MgSO(4).7H(2)O 1.50, (NH(4))(2)SO(4) 2.46, KH(2)PO(4) 0.86, K(2)HPO(4) 1.11 and Na(2)SO(4) 1.72. An overall 16% and 35% increase in antibiotic activity were obtained as compared with mean observed response (283.7U/ml) at zero level of all variables and YSG medium.     

Conversion of dechlorodauricumine into chlorinated alkaloids in Menispermum dauricum root culture

(15)N-Labeled dechlorodauricumine and dechloroacutumine were isolated from Menispermum dauricum roots cultured in a chloride-deficient medium, in which nitrogen-containing macro-components K(14)NO(3) and ((14)NH(4))(2)SO(4) were replaced by K(15)NO(3) and ((15)NH(4))(2)SO(4), respectively. These (15)N-labeled substrates were supplied independently to the roots cultured in a chloride-enriched medium. LC-ESI-MS analysis of alkaloids extracted from the roots, harvested 5 and 10 days after administering the (15)N-labeled substrates, revealed that the (15)N derived from dechlorodauricumine was much more effectively incorporated into chlorinated alkaloids than that derived from dechloroacutumine. These findings suggest that dechlorodauricumine is the principal precursor of the chlorinated alkaloids produced by M. dauricum roots.     

Diaminopimelate decarboxylase of sporulating bacteria

The meso-diaminopimelate (DAP) decarboxylase of Bacillus licheniformis, a pyridoxal phosphate-requiring enzyme, was stabilized in vitro by 0.15 m sodium phosphate buffer (pH 7.0) containing 1 mm 2,3-dimercaptopropan-1-ol, 100 mug of pyridoxal phosphate per ml, and 3 mm DAP. When the meso-DAP concentration was varied, the enzyme in cell-free extracts of B. licheniformis exhibited Michaelis-Menten kinetics. Pyridoxal phosphate was the only pyridoxine derivative which acted as a cofactor. The enzyme was subject to both inhibition and repression by l-lysine. The inhibitory effect of lysine was on the K(m) (meso-DAP). A maximum repression of about 20% was obtained. No significant inhibition or activation was produced by cadaverine, dipicolinic acid, phenylalanine, pyruvate, ethylenediamine-tetraacetate, adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate. When B. licheniformis was grown in an ammonium lactate-glucose-salts medium, an increase in DAP decarboxylase specific activity occurred during cellular growth with a maximal specific activity at the end of the exponential phase. As soon as growth ceased, the specific activity of the enzyme decreased to approximately one-half of the maximal specific activity and remained at this level thereafter. When B. cereus was grown in complex media, there was an increase in DAP decarboxylase specific activity up to the end of the exponential phase. Thereafter, the specific activity decreased to a nondetectable level in 4 hr. Dipicolinic acid synthesis was first detected 15 min later and was essentially complete after an additional 2.5 hr. The significance of the disappearance of DAP decarboxylase in B. cereus was discussed with regard to control of dipicolinic acid and spore mucopeptide biosynthesis.     

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing 相似文献   

Dehydrogenases reducing NAD+ in the presence of D (-) 3- phosphoglycerate in mycobacteria (BCG, H37Ra, M. phlei)]

Referring to the elution volume on a Sephadex G-150 column only one specific peak is obtained, the same for the BCG, H37Ra and Mycobacterium phlei strains grown on Sauton synthetic medium. Some properties of these partially purified dehydrogenases are studied (conservation and dialysis in media of different salt concentrations, equilibrium constant, Km, heat stability). All enzyme preparations from tubercle bacilli (BCG, H37Ra) are readily inactivated by heat and are very unstable in solutions of low ionic strength. In contrast, under the same experimental conditions, all enzyme samples from M. phlei are, comparatively, much more stable towards these factors [heat, salt (potassium phosphate, NaCl) concentration].     

Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01

The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH(4))(2)SO(4), MgSO(4).7H(2)O and initial pH on chitosanase production was noted with Plackett-Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0g/L (NH(4))(2)SO(4), 1.3g/L MgSO(4) and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6U/mL to 118U/mL.     

The Prevalence in Canada of Drug-Resistant Tubercle Bacilli in Newly Discovered Untreated Patients with Tuberculosis

During the period February 1963 to September 1964, the incidence of strains of tubercle bacilli excreted by newly diagnosed, previously untreated patients with tuberculosis in Canada that were resistant to the major antituberculosis drugs, viz. streptomycin, para-aminosalicylic acid and isoniazid, was 4.8%. Resistance to a single drug (3.8%) was observed more commonly than was resistance to two drugs (0.9%) or to all three drugs (0.3%), and the drug most frequently involved in this regard was streptomycin (1.8%). Data for the province of Ontario were almost the same as those for Canada. These data are compared with results of surveys in other countries and it is concluded that while drug-resistant tubercle bacilli do not constitute a major problem in Canada at present, awareness of this potential hazard should be maintained.     

Influence of nutritional and environmental factors on ethanol and endopolygalacturonase co-production by Kluyveromyces marxianus CCEBI 2011

Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianus CCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcane juice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31 oC; total reducing sugars (TRS), 125 g/l; (NH(4))(2)SO(4), 2.48 g/l; (NH(4))(2)HPO(4), 2.73 g/l; CaCl(2), 0.33 g/l and MgSO(4)·7H(2)O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH(4))(2)SO(4) supplementation were the most significant factors influencing the co-production process.     

The determination of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine in serum and urine

1. A solvent system was devised for the extraction of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine from serum and urine. 2. Specific chemical and fluorimetric methods were developed for the determination of the extracted isoniazid and acetylisoniazid, and chemical methods for the determination of monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine. 3. When applied to serum, these methods were capable of measuring concentrations of down to about 0.005mug of isoniazid/ml, 0.05mug of acetylisoniazid/ml, 0.2mug of monoacetylhydrazine/ml, 0.2mug of diacetylhydrazine/ml, 0.02mug of isonicotinic acid/ml and 0.1mug of isonicotinylglycine/ml. 4. In urine, these methods were capable of measuring concentrations of down to about 0.05mug of isoniazid/ml, 0.2mug of acetylisoniazid/ml, 1mug of diacetylhydrazine/ml, 0.1mug of isonicotinic acid/ml and 0.2mug of isonicotinylglycine/ml. 5. The stability of these compounds was studied in serum and urine and a method devised to decrease their decomposition in serum.     

Production of rosamicin: improvement of synthetic medium

Rosamicin is one of the important macrolide antibiotics that has clinical efficacy and broad-spectrum antibacterial activity. Using a mutant strain of Micromonospora rosaria (NRRL 3718), a chemically defined medium was developed, and some fermentation conditions that are important to rosamicin biosynthesis were optimized to achieve rosamicin productivity of 230 mug/ml. Soluble starch and l-asparagine were found to be the best carbon and nitrogen sources, and a stimulative effect of magnesium and zinc ions was also found. The medium developed contains: soluble starch, 4%; l-asparagine, 0.15%; K(2)HPO(4), 0.075%; CaCO(3), 0.6%; MgSO(4) . 7H(2)O, 0.05%; FeSO(4) . 7H(2)O, 10 M; CuSO(4) . 5H(2)O, 10 M; ZnSO(4) . 7H(2)O, 10 M; and MnSO(4) . (4-6)H(2)O, 10 M. The required air supply was about 40 mmol of O(2) liter . h . atm, and the favorable culture temperature was 28 to 29 degrees C.     

Production of l-Glutamine by a Penicillin-Resistant Mutant of Flavobacterium rigense

To establish a practical method for the fermentative production of l-glutamine, cultural conditions for the accumulation of a large amounts of l-glutamine were investigated by using Flavobacterium rigense 703, which was previously reported by us as a l-glutamine-producing mutant. As a result, a yield of 25 mg of l-glutamine per ml was obtained after a 48-h cultivation in a medium containing glucose, yeast extract, (NH(4))(2)-fumarate, KH(2)PO(4), K(2)HPO(4), MgSO(4).7H(2)O, and CaCO(3) (pH 6.4). Accumulation of l-glutamine was dependent upon the concentration of (NH(4))(2)-fumarate, and a suboptimum growth at a relatively high concentration of (NH(4))(2)-fumarate was essential for the maximum production of l-glutamine. At the optimum conditions, glutamic acid was formed as a by-product at a concentration of less than 1 mg/ml, but accumulation of the other amino acids was negligible. The product was isolated from the culture broth and readily purified by anion-exchange chromatography. The pure crystals of l-glutamine obtained in an 80% yield were optically and chromatographically pure.