Isolation of the new antibiotic variamycin B from the biosynthetic products of a Streptomyces olivovariabilis sp. nov. culture

Variamycin B, a new antitumor antibiotic was isolated from metabolic products of Streptomyces olivovariabilis sp. nov. The empirical formula of variamycin B is C52H76O24, the melting point is 163-165 degrees C (dec.), (alpha)20D--30 +/- 2 degrees (C 0.5, alcohol), the UV-spectrum: lambda max 230, 279, 317, 330, 415 (lg E 4.27, 4.6, 3.83, 3.75, 3.95), IR-spectrum: 1080, 1170, 1380, 1570, 1640, 1720, 3400 cm-1. Variamycin B is classified as belonging to the group of antibiotic analogs of aureolic acid. Chromomycinone, tetrazide and residues of two 2,6-didesoxysugars, i.e. olivose and variose were obtained as a result of complete and partial acid degradation of variamycin B. It was shown with the data of quantitative analysis of the hydrolysates obtained on complete hydrolysis of variamycin B that the ratio between the residues of variose and olivose in the antibiotic molecule was 1 : 4. Oxidation of variamycin B with Fremi salt resulted in formation of chinone having chromomycinone-chinone, 2 residues of olivose and 1 residue of variose in its composition. Investigation of the products of partial hydrolysis provided isolation of a substance having aglicone-chromomycenone and 4 residues of olivose in its composition. A partial structure of variamycin B is proposed.     

Action of aureolic acid and dactinomycin group antibiotics on human brain tumors in culture]

The cytotoxic effect of antitumour antibiotics, such as dactinomycin, mithramycin, variamycin and olivomycin on the cells of the human brain tumours (multiform glyoblastoma, arachnoidendotelioma and astrocytoma) grown by the method of the primary plasmic culture was studied. Dactinomycin was superior to the antibiotics of the aureolic acid group in the rate and level of the cytotoxic effect on the tumour cells: 76 per cent of the above tumours were sensitive to dactinomycin, 56 per cent to mithramycin and 52 per cent to variamycin and olivomycin. Among the total number of the tumours sensitive to the drugs the number of the highly sensitive tumours amounted to 57.9 per cent for dactinomycin and 30.8--38.5 per cent for the antibiotics of the aureolic acid group. Definite differences in the efficiency of the antibiotics of the aureolic acid group with respect to different types of the brain tumours were observed.     

Study of the distribution of C14-variamycin and C14-mitramycim after intravenous administration to mice]

Distribution of antitumor antibiotics, i.e. C14-variamycin and C14-mitramycin in the organs of albino mice after their intravenous administration in single doses was studied. Similarity in the distribution dynamics of both the antibiotics with respect to the animal organs was found. However, the level of variamycin as compared to that of mitramycin was much higher in the liver and especially the spleen. In the experiments with variamycin the radioactivity of the kidney tissue decreased more rapidly than in the experiments with mitramycin. Chromatographic analysis of the urine of the mice treated with C14-variamycin was performed. The labeled Variamycin was detected in the animal urine within 48 hours from the moment of the antibiotic administration. Its portion in the total amount of the radioactive products in the urine was 30 to 40% at various stages of the study.     

Long-term reversible suppression of oestrus in bitches with nafarelin acetate, a potent LHRH agonist

Adult cyclic beagle bitches were treated for up to 18 months with nafarelin acetate via subcutaneously implanted osmotic pumps, starting during the first week of a pro-oestrous vaginal discharge. The imminent ovulation appeared to be unaffected by treatment, but doses of 8 or 32 micrograms analogue/day reduced the integrated luteal progesterone values. No new oestrus was detected in 3 bitches during 18 months of treatment with 32 micrograms/day, which resulted in mean plasma levels of 0.4 ng analogue/ml. A return to oestrus was observed in all 3 bitches between 3 and 18 weeks after cessation of treatment: 2 of the bitches mated at those times and produced normal litters. Another 2 bitches were similarly treated with 32 micrograms analogue/day; they were mated at the oestrus at start of treatment and dosing was continued for about 63 days. One of the bitches conceived and produced a normal litter. Nafarelin acetate treatment begun during anoestrus resulted in an induced heat 1-2 weeks after the start of treatment. The induced heat consisted of pro-oestrous vaginal discharge, oestrous vaginal cytology, and ovulation (judged by increased circulating levels of progesterone). Three bitches mated at the induced heat and treated for the normal duration of gestation did not litter. Nafarelin treatment of 3 bitches before puberty did not induce signs of oestrus and prevented the occurrence of oestrus through 18 months of treatment. The first oestrus in these bitches occurred 3.5-4 months after cessation of treatment, but mating at that time did not result in pregnancy. These studies have established the feasibility of and dosage requirement for the use of the LHRH agonist as a contraceptive in the bitch.     

Development and validation of a HPLC method for the quantitation of ochratoxins in plasma and raw milk

A HPLC method with improved sensitivity for the determination of ochratoxins (OT) A, B and alpha in plasma and milk was developed. Plasma analysis involved a simple liquid-liquid extraction with chloroform; while for milk, an additional immunoaffinity clean-up step was necessary. The method showed a good linearity (r(2)>0.999). The limit of quantification (LOQ) of OTA was 5 and 200 ng/l for milk and plasma, respectively. Average recovery was 89% in both matrices, except for OTalpha in milk that was only 18% due to poor immunoaffinity binding. OT remained stable in -20 degrees C stored samples; OTA concentration in plasma and milk did not change after 8 and 18 months of storage, respectively. The developed method has been applied to contaminated plasma and milk samples obtained from dairy ewes fed with ochratoxin-contaminated feed.     

Efficacy of some methods and media for detecting and enumerating Campylobacter jejuni in frozen chicken meat

Four of five strains of Campylobacter jejuni survived in chicken meat stored at -18 degrees C for 12 months. Direct plating of samples was superior to the most probable number technique for enumerating C. jejuni. Enrichment culture using the Doyle & Roman enrichment method resulted in the highest rates of detection. Packaging under an atmosphere of CO2 did not substantially influence survival.     

Semiquantitative Oropharyngeal Culture Technique

A semiquantitative method for determining the concentration of organisms constituting the normal oropharyngeal flora has been developed. Eleven species of organisms were isolated from the 18 subjects studied. The concentration of organisms in multiple samples, taken at 5-min intervals, was quite similar. The concentration of organisms increased slightly at the end of the day. Obtaining specimens on different days of the week did not appreciably affect the concentration or kinds of organisms. Eating had only a minimal effect on the oropharyngeal flora, but brushing teeth reduced the concentration of organisms substantially. When specimens were obtained 6 months after the initial specimens, the concentration of organisms remained the same but the species of organisms isolated varied considerably. The gargle method was compared to a swab method and proved to be superior. This method of obtaining oropharyngeal culture specimens is reliable and useful as a means of monitoring the normal oropharyngeal flora.     

Age and Strain Comparisons of Neurotransmitter Synthetic Enzyme Activities in the Mouse

Activities of the neurotransmitter synthetic enzymes, choline acetyltransferase (EC 2.3.1.6; ChAT), glutamic acid decarboxylase (EC 4.1.1.15; GAD), and tyrosine hydroxylase (EC 1.14.3.2; TH), were assayed in four brain regions of A/J and C57BL/6J mice at three ages (4, 18, and 24 months). The brain regions assayed were the fronto-parietal cortex, hippocampus, striatum, and cerebellum. Strain effects: In some brain regions, at several ages, ChAT activity did not differ among the two strains. However, ChAT was higher in the C57BL/6J strain in the cortex at 18 months, the hippocampus at 18 and 24 months, the striatum at 24 months, and the cerebellum at 4 months. The reverse was true in the cerebellum at 24 months, where ChAT was higher in A/J mice. GAD activity in C57BL/6J mice compared to that of A/J mice was higher in the striatum and cortex, and lower in the hippocampus and cerebellum. TH activities in all four regions were generally higher in C57BL/6J mice than in A/J mice. Age effects: Age differences in enzyme activities varied with the genetic strain. ChAT activity generally was higher in brain regions of older mice of both strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of some methods and media for detecting and enumerating Campylobacter jejuni in frozen chicken meat

Four of five strains of Campylobacter jejuni survived in chicken meat stored at — 18C for 12 months. Direct plating of samples was superior to the most probable number technique for enumerating C. jejuni. Enrichment culture using the Doyle & Roman enrichment method resulted in the highest rates of detection. Packaging under an atmosphere of CO₂ did not substantially influence survival.     

Factors Associated With Insulin Pump Discontinuation in Adults With Diabetes: A Time-to-Invent Analysis and Prediction Model

ObjectiveInsulin pump discontinuation has mostly been studied in children and adolescents living with diabetes. We aimed to assess the rate of insulin pump continuation in a population of adult patients with diabetes, at 18 months after initiation; determine the factors associated with pump discontinuation; and develop a simple prediction model.MethodsThis single-center, retrospective study included all adult patients with type 1 diabetes or type 2 diabetes who started insulin pump treatment between January 2015 and June 2018. The exclusion criteria were pregnancy, short-term pregnancy plans, and insulin pump discontinuation within the previous 6 months. The probability of insulin pump continuation after 18 months was estimated using the Kaplan-Meier method. Factors associated with insulin pump discontinuation were studied using a Cox regression model, and an exponential model was built for prediction purposes.ResultsThe study included 315 patients. The mean age was 41 years, the mean duration of diabetes was 16 years, 50% were men, 74% had type 1 diabetes, and the mean hemoglobin A1c level was 9.1% (76 mmol/mol). After 18 months, the rate of insulin pump continuation was 0.80 (95% Confidence Interval (CI), 0.76-0.85). By multivariate analysis, the occurrence of severe hypoglycemia in the previous year was associated with insulin pump discontinuation (hazard ratio, 2.42; 95% CI, 1.30-4.51), while other factors did not reach statistical significance.ConclusionInsulin pump discontinuation occurred in 20% of patients at 18 months after initiation and was mainly associated with a recent history of severe hypoglycemia. The type of diabetes and glycemic control at baseline were not associated with treatment discontinuation.     

Long-term retention of internal elastomer tags in a wild population of painted crayfish (Panulirus versicolor [Latreille]) on the Great Barrier Reef

Many crustaceans are highly exploited fisheries species, but determining the population parameters of these animals via mark-recapture techniques is problematic, primarily due to tag-loss during moulting of the exoskeleton. Recently developed internal elastomer tags may overcome this challenging problem, since they are completely encased in the tissues beneath the exoskeleton. In this study, we evaluated the effectiveness of internal elastomer tags in a wild population of painted crayfish (Panulirus versicolor) over an 18-month period. Seventy animals were double-tagged in the abdominal musculature, with individuality obtained using different combinations of tag colour and tag location. Forty individuals were recaptured after 6, 12 and (or) 18 months, giving an overall recapture rate of 57%. Annual tag retention was estimated to be 98%, since only one instance of tag-loss was observed, despite numerous episodes of moulting among tagged individuals. The majority of tags (95%) were easily visible, even after 18 months at liberty. Nonetheless, small reductions in tag condition were observed after the first six months (mostly due to fading and [or] fragmentation), but this did not interfere with overall tag readability. Tag condition did not decline in the following 12 months, nor did it vary with gender or tag location. It is concluded that internal elastomer tags are an effective method for marking wild crayfish over long-term periods. Given the durability of elastomer, and the capacity for individual identification of large numbers of animals, such tags may be suitable for commercial-scale fishery applications.     

Impact of community-directed treatment on soil transmitted helminth infections in children aged 12 to 59 months in Mazabuka District, Zambia

This study assessed the impact of adding community-directed treatment (ComDT) to the routine health facility (HF)-based treatment on prevalence and intensity of soil transmitted helminth (STH) infections among children aged 12 to 59 months. Repeated cross-sectional surveys were conducted among randomly selected children of this age group from the intervention area (HF+ComDT area) and the comparison area (HF area) at baseline (n=986), 12 months (n=796) and 18 months (n=788) follow-up. The prevalence of Ascaris lumbricoides was significantly higher in the HF+ComDT as compared to the HF area at baseline (P=0·048), but not at 12 and 18 months follow-up. At baseline the HF+ComDT area had significantly higher intensities of A. lumbricoides compared to the HF area (P<0·001), but not at 12 and 18 months follow-ups. Prevalence and intensity of hookworm did not differ significantly between treatment arms at any time. Analysis of trends showed a significant decrease in prevalence of A. lumbricoides and hookworm in the HF+ComDT area (P<0·001), of hookworm in the HF area (P<0·05), but not of A. lumbricoides in the HF area. It is concluded that the ComDT approach generally enhanced the treatment effect among under-five year children and that this alternative approach may also have advantages in other geographical settings.     

Analytical method for the determination of urinary 3-phenoxybenzoic acid in subjects occupationally exposed to pyrethroid insecticides

The determination of urinary 3-phenoxybenzoic acid enables exposure to pyrethroid insecticides to be evaluated. A method for the quantitative determination of this metabolite in urine is described. The compound and the internal standard (2-phenoxybenzoic acid) are derivatized with pentafluorobenzylbromide and transformed into pentafluorobenzyl esters, which are determined by gas chromatography with an intermediate polarity capillary column and an electron-capture detector. Before GC analysis, the urinary extracts are purified on LC-Si SPE columns. The proposed method has a detection limit of 0.5 μg/l and a mean recovery of 91.3%. The coefficient of variation of the analytical procedure, evaluated at a concentration of 24.96 μg/l, was 9.58%. Storage of the urine samples for 3 months at −18°C did not lead to significant changes in the concentration of analyte. The method was tested analysing the urine of a farm worker with symptoms of pyrethroid poisoning, occupationally exposed to esfenvalerate.     

Fluctuations in peripheral serum testosterone levels within a day, with age and by sexual stimulation in male beagle dogs bred indoors

Fluctuations in serum levels of testosterone (T) within a day, both with age and as a result of sexual stimulation, were examined in male beagle dogs. Eighty male dogs aged between 3 months and 16 years and bred in our laboratory were used under strictly controlled breeding conditions (temperature: 22 +/- 1 degree C, relative humidity: 55 +/- 5%, lighting time: 8:00 a.m.-8:00 p.m.). The level of T was measured by an RIA method. In order to examine the fluctuation in T level within a day, blood samples were obtained at 0:00, 6:00, 12:00 and 18:00 in each of five dogs aged 1.7 and 2.1 years. T levels fluctuated with a regular pattern that was lowest at 12:00, and increased to a peak at 18:00-6:00, thereafter decreasing until 12:00. In order to examine the change in T level with age, blood samples were obtained at 9:00, 12:00 and 16:00 from 70 dogs aged between 3 months and 16 years. The regular diurnal pattern of T level was usually seen, and the levels at 12:00 were always low and did not fluctuate at any age except for 6 months, and 13, 14 and 16 years. The T level at 9:00 increased to reach a peak at 4 years, whereas that at 16:00 did so at 2 years. T levels at 9:00 were significantly higher at 4-12 and 14 years than at 3 months, and were higher at 4 years than at 9 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface-active properties of antibiotics

The critical concentrations of the mycella formation of novobiocin, mithramycin, variamycin, erythromycin, oleandomycin and lincomycin were determined with two methods by changes in the isotherms of the surface tension and in the maximum absorption of rodamine due to the antibiotic concentrations. The results obtained with the two methods were comparable.     

Development and validation of a high-performance liquid chromatography assay for the quantitative determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma

A new, simple, reproducible and reliable high-performance liquid chromatography method with ultraviolet absorbance detection at 240 nm was developed and validated for the determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma. The method was based upon solid-phase (C₁₈) extraction of plasma after addition of 17β-hydroxy-3β-methoxyandrost-5-en-7-one as internal standard. Using 1 ml of plasma for extraction, the detection limit of the assay was 3 ng/ml. The standard curve was linear over the concentration range 10–1000 ng/ml. Stored at −20°C for about 4 months at various concentrations in plasma, 7-oxo-dehydroepiandrosterone-3β-sulfate did not reveal any appreciable degradation. Also included herein is a method for the simultaneous detection and determination of 7-oxo-dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone-3β-acetate in plasma.     

Impact on lipoprotein profile after long-term testosterone replacement in hypogonadal men.

Testosterone serum levels may influence the lipoprotein metabolism and possibly atherogenic risk. Our aim was to investigate the effects of long-term testosterone supplementation in hypogonadal men on multiple lipoprotein markers. 18 Hypogonadal men were studied before and after 3, 6, and 18 (n = 7) months of treatment with testosterone enanthate. During treatment, serum testosterone and estradiol increased, reaching normal levels (p < 0.0001 and 0.003, respectively). This was associated with a decrease in HDL cholesterol (from 1.40 +/- 0.10 mmol/l to 1.22 +/- 0.08 mmol/l, p < 0.001) after six months at the expense of HDL2 cholesterol (p < 0.01), as well as apoprotein A1 (from 139 +/- 3.4 mg/dl to 126 +/- 3.0 mg/dl, p < 0.005). Hepatic lipase activity increased (p < 0.05) and correlated positively with testosterone (r = 0.56, p < 0.02) and negatively with HDL cholesterol (r = - 0.58, p < 0.02). Total and LDL cholesterol, triglycerides, and apoprotein B did not increase. Among the seven patients who completed 18 months of treatment, triglycerides, total cholesterol, LDL and HDL cholesterol, as well as total cholesterol/HDL cholesterol ratio values did not differ from baseline while apoprotein A1 (p < 0.03) and HDL cholesterol (p < 0.015) remained decreased and hepatic lipase unchanged. Restoration of testosterone levels in hypogonadal men in this study did not reveal unfavorable changes based on total cholesterol/HDL cholesterol and LDL cholesterol/apoprotein B ratios, which are both atherogenic risk markers. Whether the changes in light of lipoprotein metabolism will adversely influence cardiovascular risk over time remains to be determined.     

Temporary increase in chromosome breakage in an infant prenatally exposed to lead

Summary An infant exposed to high levels of lead in utero was found to have increased numbers of cells with chromosome breaks in blood samples obtained at 6 weeks and 3 months of life. Later samples did not show significant abnormality. Physical and neurological examinations of the patient up to 18 months of age gave results within normal limits.     

Parameters of the toxic action of antibiotic derivatives of aureolic acid in acute and subchronic experiments]

The parameters of the lethal effect of aureolic acid derivatives, such as mithramycin, variamycin and olivomycin were studied on mice, rats and rabbits. As for the most of the administration routes the first two drugs were characterized by irregular distribution of resistance to thier lethal effect, which was mathematically expressed by polymodality of the dose-response curve. The above drugs were characterized by cumulative properties. The toxicity parameters depended on the animal species and administration route.     

Localization and age-related changes in cytochrome P450 expression in APA hamster livers.

To establish the baseline data, age-related changes and the regional expression of the hepatic P450 isozymes in Syrian hamsters of the APA strain at 3, 6, 12, 18 months old were examined by immunological techniques. Immunohistochemical analysis of liver serial sections revealed that the midzonal and perivenous regions (zones 2 and 3, respectively) were stained with the anti-rat CYP1A1/2, 2B1/2 and 2E1 antibodies. These three antibodies most intensely stained the hepatocytes around the central vein. An anti-rat CYP3A2 staining section had a staining pattern with equally intense reactions in zones 2 and 3. On the other hand, CYP2C6, 2C11 and 4A1 were distributed diffusely throughout the hepatic acinus. There was no age-related difference in the expression pattern of any of the P450 isozymes examined. Total P450 content had a peak at 6 months of age and decreased to 60% of that level thereafter. Western-blot analysis revealed that the peak expressions of the isozymes detected with anti-rat CYP1A1/2, 2C6, 2E1 and 3A2 antibodies were observed in 6-month-old hamsters and declined in older ones. The CYP2B and 2C11 content reached the maximum at the age of 6 months and maintained almost the same level thereafter. The CYP4A level did not change from 3 to 6 months, and then declined to about 40% of the younger level at 12 and 18 months of age. These results suggest that the hepatic P450 isozymes of APA hamsters have region-specific expressions and most isozymes have their peaks of expression at 6 months of age, which differs from the patterns for rat P450.