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Enzymatic formation of the bisfuran structure in aflatoxin biosynthesis.

A relatively stable enzyme system that converts versiconal hemiacetal acetate to versicolorin A was isolated from the soluble fraction of the homogenized cells of Aspergillus parasiticus ATCC 15517. The cell-free preparation did not require oxygen or oxidized nicotinamide adenine dinucleotide phosphate for activity, nor did it require dithiothreitol, polyclar (polyvinyl pyrrolidone), or glycerol for stabilization of activity. It was susceptible to inhibition by dichlorvos and cysteine. Isotope tracer studies revealed involvement of several intermediates in the conversion of versiconal hemiacetal acetate to versicolorin A. These findings confirm the biogenetic relationship of versiconal hemiacetal acetate and versicolorin A, and they confirm that the bisfuran ring structure in aflatoxins and related fungal metabolites is derived from the hemiacetal structure of versiconal hemiacetal acetate.

Aflatoxins↗

Thymocytotoxic autoantibodies found in mice infected with Schistosoma japonicum.

Thymocytotoxic autoantibodies were demonstrated in sera of C3H/HeJms, C57BL/6J, and ddY mice infected with 50 cercariae of Schistosoma japonicum, using C57BL/6J thymocytes as target cells in the trypan blue dye exclusion test. Kinetic study revealed that thymocytotoxic activity began to increase at week 6 of infection, reached a maximum at 8 weeks, and thereafter decreased gradually. Thymocytotoxic antibodies had an optimal reactivity at 4 degrees C and were sensitive to 2-mercaptoethanol treatment, suggesting that they were immunoglobulin M in nature. The cytotoxicity was completely abolished by absorption with C57BL/6J thymocytes but not with S. japonicum parasites or eggs. The antigen reacting with thymocytotoxic antibodies was found in the thymus, brain, spleen, and, to a lesser extent, kidney and liver. In parallel with the appearance of thymocytotoxic antibodies, the increase of background plaque-forming cells to trinitrophenyl, polyvinyl pyrrolidone, and sheep erythrocytes in the spleen of S. japonicum-infected mice suggested that te induction of thymocytotoxic antibodies may be the consequence of polyclonal B-lymphocyte stimulation by the infection.

Animals↗

Distribution of calcium and other elements in cryosectioned Bacillus cereus T spores, determined by high-resolution scanning electron probe x-ray microanalysis.

The distribution of a number of key elements in Bacillus cereus T spores was determined by high-resolution scanning electron probe X-ray microanalysis. To circumvent the redistribution of soluble or weakly bound elements, freeze-dried cryosections of spores, which had been rapidly frozen in 50% aqueous polyvinyl pyrrolidone, were employed. The sections were examined by using a modified Philips EM400 electron microscope fitted with a field emission gun, scanning transmission electron microscopy attachment, and a computer-linked energy-dispersive X-ray microanalysis system. X-ray maps for selected elements and the corresponding electron image were produced simultaneously by scanning the cryosections with a fine electron beam in a raster pattern, using the scanning transmission electron microscopy attachment. The results indicated that almost all of the calcium, magnesium, and manganese, together with most of the phosphorus, was located in the core region. An unexpectedly high concentration of silicon was found in the cortex/coat layer. Granules containing high concentrations of calcium, manganese, and phosphorus were demonstrated in spores containing reduced levels of dipicolinic acid. Spot mode analyses, in which a stationary beam was located over the region of interest in the spore cryosection, confirmed the results obtained with the scanning mode and also provided a more accurate quantitation of the elemental concentrations on a dry weight bases.

Bacillus cereus↗

Nutrition of Leptospira pomona. 1. A chemically defined substitute for rabbit serum ultrafiltrate.

Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. I. A chemically defined substitute for rabbit serum ultrafiltrate. J. Bacteriol. 83:668-672. 1962.-Poor growth of Leptospira pomona, strain Wickard, in a medium containing exhaustively dialyzed rabbit serum was corrected by addition of rabbit serum ultrafiltrate. A dialyzed serum medium was used to investigate the nutritional qualities of various compounds when tested as substitutes for the ultrafiltrate fraction. l-Asparagine was the only amino acid that markedly stimulated growth. l-Glutamine was active only when autoclaved (converted to the ammonium salt of pyrrolidone carboxylic acid), and NH(4)Cl and urea satisfactorily replaced l-asparagine. Results indicated that amino acids functioned primarily as a nitrogen source in this medium. Thiamine was the only vitamin required for growth. The lag phase was usually decreased by 1 day with NaHCO(3).A medium composed of 0.02 m phospate buffer (pH 7.4), 15% dialyzed rabbit serum, thiamine (5 mug/ml), and NH(4)Cl (10(-3)m) supported growth equivalent to that obtained with undialyzed rabbit serum medium. L. pomona was transferred six times, in this medium and seven other serotypes were transferred three times, without any decrease in amount of growth. Growth of L. pomona was initiated with 20 organisms per ml.

Animals↗

Bacteremia due to Moraxella atlantae in a cancer patient.

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.

Adenocarcinoma↗

[Promoters of genes MTHFR from patients with hyperhomocysteinemia and PTEN from patients with malignant and benign endometrial and ovarian tumors].

Mutational changes in the promoter regions of MTHFR genes from patients with hyperhomocysteinemia and PTEN genes from patients with endometrial and ovarian tumors were studied. An increased level of homocysteine was found in a part of the patients with a heterozygous C677T mutation in the MTHFR gene, although a moderate hyperhomocysteinemia is usually associated with homozygous mutation. We hypothesized that, in this case, the allele lacking the C677T mutation may be inactivated by the promoter mutation. The sequencing of both DNA strands of the minimal promoter region of the MTHFR gene in ten patients did not reveal any mutation, which implied another mechanism of the development of hyperhomocysteinemia in these patients. A PCR analysis of the minimal promoter region of the tumor suppressor PTEN in the presence of 2-pyrrolidone in 101 patients from Moscow clinics revealed changes in it in patients with endometrial (56%) or ovarian (29%) cancer, as well as in patients with endometrial hyperplasia and benign ovarian tumors (34.6 and 29%, respectively). It was presumed that the found PTEN gene promoters may arise from epigenetic alterations (erroneous methylation) or may (more rarely) be induced by mutations. As a result of the studies, new molecular markers associated with endometrial and ovarian tumors were revealed and a simple and effective method of detection of these markers was developed.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

Extracting DNA from submerged pine wood.

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

Cell Nucleus↗

Specificity of gamma-glutamyl cyclotransferase.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.

Acyltransferases↗

The thermoregulatory mechanism of melatonin-induced hypothermia in chicken.

The involvement of melatonin (Mel) in body temperature (Tb) regulation was studied in White Leghorn layers. In experiment 1, 35 hens were injected intraperitoneally with seven doses of Mel (0, 5, 10, 20, 40, 80, or 160 mg Mel/kg body wt) dissolved in ethanol. Within 1 h, Mel had caused a dose-dependent reduction in Tb. To eliminate a possible vehicle effect, 0, 80, and 160 mg/kg body wt Mel dissolved in N-methyl-2-pyrrolidone (NMP) was injected. NMP had no effect on Tb, with Mel again causing a dose-dependent hypothermia. In experiment 2 (n = 30), Mel injected before exposure of layers to heat reduced Tb and prevented heat-induced hyperthermia. Injection after heat stress had begun did not prevent hyperthermia. Under cold stress, Mel induced hypothermia, which was not observed in controls. In experiment 3 (n = 12), Mel injection reduced Tb and increased metatarsal and comb temperatures (but not feathered-skin temperature), respiratory rate, and evaporative water loss. Heart rate rose and then declined, and blood pressure increased 1 h after Mel injection. Heat production rose slightly during the first hour, then decreased in parallel to the Tb decline. We conclude that pharmacological doses of Mel induce hypothermia in hens by increasing nonevaporative skin heat losses and slightly increasing respiratory evaporation.

Animals↗

Production of Prednisolone by Pseudomonas oleovorans Cells Incorporated Into PVP/PEO Radiation Crosslinked Hydrogels.

In order to rise the yield of prednisolone from hydrocortisone, the Pseudomonas oleovorans cells were entrapped into radiation crosslinked poly (vinyl pyrrolidone)/poly(ethylene oxide) (PVP/PEO) hydrogel of different gel contents. The factors affecting the gel content and swelling behavior of the polymeric gel, such as polymer composition, polymer blend concentration, and irradiation doses, were investigated. The formation of gels having a good strength with the ability to retain a desirable amount of water in their three-dimensional network can be achieved by using PVP/PEO copolymer of composition $(90:10)$ and concentration of 15% prepared at 20 kGy irradiation dose. At these conditions the prepared hydrogel is considered the most favorable one that gave the highest hydrocortisone bioconversion and prednisolone yield, 81% and 62.8%, respectively. The improvement of prednisolone yield was also achieved by increasing substrate concentration. Maximum hydrocortisone bioconversion (86.44) was obtained at 18 hours by using substrate concentration of 30 mg. Reusability of immobilized Pseudomonas oleovorans entrapped into PVP/PEO copolymer hydrogel was studied. The results indicated that the transformation capacity of hydrocortisone to prednisolone highly increased by the repeated use of copolymer for 4 times. This was accompanied by an increase in prednisolone yield to 89% and the bioconversion of hydrocortisone was 98.8%.

Journal Article↗

Involvement of catecholaminergic and cholinergic mechanisms in the pulsatile release of LH in the long-term ovariectomized rat.

In the long-term ovariectomized rat the secretion of LH has a pulsatile character. In such rats no difference was observed between morning and afternoon LH secretion. The administration of phenoxybenzamine, an chi-adrenergic blocker, resulted in depressed plasma LH levels. chi-Methyl-tyrosine (chi-MT), an inhibitor of tyrosine hydroxylase had no effect on plasma LH levels, whereas bis(4methyl-1-homopiperazinil-thiocarbonil) disulphide (FLA 63), an inhibitor of dopaminic-beta-hydroxylase, induced decreased plasma LH levels and disappearance of the pulsations. The same effect was observed after the administration of apomorphine, a dopaminic receptor stimulating drug, whereas the administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966), which blocks dopamine release, significantly raised plasma LH levels. Scopolamine, a cholinergic muscarinic receptor blocking drug, had no effect on plasma LH levels, whereas mecamylamine, a cholinergic nicotine receptor blocking agent, decreased them. These results are consistent with the hypothesis that the pulsatile release of LH in the long-term ovariectomized rat is caused by the stimulating activity of adrenergic and cholinergic, probably nicotinic, systems and the inhibitory activity of a dopaminergic system.

Animals↗

Dose-dependent suppression of postcastration luteinizing hormone secretion exerted by exogenous prolactin administration in male rats: a model for studying hyperprolactinemic hypogonadism.

We examined the inhibitory effects of acute hyperprolactinemia on the postcastration rise in mean luteinizing hormone (LH) levels in adult male rats. The animals were administered purified ovine prolactin (oPRL) subcutaneously in a polyvinyl-pyrrolidone depot every 12 h for 96 h, beginning at the time of castration. oPRL suppressed postcastration LH secretion from 24 to 72 h when the effect spontaneously reversed in the face of elevated oPRL levels. oPRL suppressed postcastration LH secretion in a graded, dose-dependent fashion. The rats were administered increasing doses of oPRL and studied 48 h later, a time of maximal LH suppression. The first significant inhibition began in the high physiological range (about 200 ng/ml) and continued into the pathophysiological tumor range (about 2,500 ng/ml) of circulating oPRL levels. The highest oPRL levels markedly suppressed postcastration LH release. Autoregulatory feedback of oPRL on endogenous rat PRL secretion was examined in the dose-response study. It was striking to discover that PRL autofeedback appeared regulated as a threshold instead of a graded dose response and, more importantly, that the oPRL dose which produced the first significant suppression of LH secretion was the same dose which exerted PRL autofeedback. These findings indicate that postcastration LH secretion is inhibited by circulating PRL titers (about 200 ng/ml) which are above basal and stress-induced levels, but are within the range encountered during pregnancy, pseudopregnancy, and lactation. In addition, a common hypothalamic mechanism (perhaps dopaminergic), activated by elevated oPRL levels in this range, may inhibit both LH and rat PRL secretion.

Adrenalectomy↗

Renin granules isolated from rat kidney cortex by continuous colloidal silica (Percoll) density gradient centrifugation.

Renin granules were isolated from rat kidney cortex by a continuous polyvinyl-pyrrolidone-coated colloidal silica (Percoll) density gradient centrifugation. A major peak of renin activity was found at a density of 1.12-1.13 g/ml, and the specific activity of renin in the peak fraction was increased by approximately 70-fold, as compared with that in the kidney cortex homogenate. On the other hand, activities of other reference enzymes, such as succinate dehydrogenase, acid phosphatase and glucose-6-phosphatase, were not detectable in the peak fraction. When the extract of the peak fraction was applied to a pepstatin column, trypsin-activated renin could not be detected in the breakthrough fractions. These results indicate that renin granules of the rat kidney cortex contain only active renin.

Animals↗

Tumoricidal activity of antiseptics with assessment of cell viability in mice with severe combined immunodeficiency.

Previously, we have assessed the efficacy of different cytotoxic agents on the viability of SW620 human colonic carcinoma cells in vitro. In this study, we have investigated the tumoricidal efficacy of some antiseptic agents on SW620 human colonic carcinoma cells which were subsequently inoculated into severe combined immunodeficient (SCID) mice. An inoculum of 5 x 10(6) cells suspended in 200 microliters buffer solution was found to be the minimum number of cells needed to result in tumour growth within 8 weeks after subcutaneous injection into SCID mice. The integrity of the cells was assessed in vitro with the trypan blue exclusion test after 30 min incubation in distilled water (DW), chloramine 0.5% in DW and polyvinyl pyrrolidone iodine (PVP-I) 0.01, 0.05, 0.1 and 5% in DW. DW and PVP-I 0.01% were not tumoricidal, neither in vitro nor in vivo. In contrast, PVP-I 5% and chloramine 0.5% 'killed' all or almost all tumour cells in vitro and prevented their growth in vivo. PVP-I 0.05 and 0.1% were less effective in vitro than 5%, but could prevent in vivo proliferation unless an adjustment of the residual number of viable tumour cells was performed. These data indicate the importance of the amount of the tumour inoculum and hence the need to use a maximally effective 'killing' agent.

Animals↗

Studies on glomerular permeability using inert polymers.

Dextrans and other macromolecular inert polymers such as polyvinyl pyrrolidone fulfil all the criteria of an ideal test material for measuring glomerular permeability. Following a single intravenous injection of radioactively labelled polydisperse material serial plasma and urine samples can be analysed by gel filtration chromatography and the polymer fractionated into its constituent molecular sizes. Polymer of molecular size 24 A is capable of passing through the glomerulus as readily as water while material larger than 60 A is affectively excluded from the glomerular filtrate and one can determine the clearance of 20--30 intermediate molecular sized fractions. The accumulated data can be analysed by a number of different theoretical mathematical models; furthermore the technique can be employed in the study of proteinuria induced by vasoactive amines such as renin or angiotensin.

Animals↗

Viscoelasticity of radiation-formed PVA/PVP hydrogel.

The dynamic rheological behaviour of gamma-irradiated 12.8 wt% poly(vinyl alcohol) (PVA), 12.8 wt% poly(vinyl pyrrolidone) (PVP), and a blend of 8 wt% PVA and 4.8 wt% PVP aqueous solutions have been studied pre- and post-gelation. The non-irradiated solutions displayed rheological behaviour typical of dilute to semi-dilute polymer solutions, with the complex viscosity being independent of the frequency and shear rate (i.e. Newtonian behaviour) over the range of frequencies tested and the loss modulus G"(omega) and storage modulus G'(omega) being nearly proportional to omega and omega2, respectively. After a set of doses of gamma-radiation, the magnitudes of the dynamic moduli G'(omega) and G"(omega) increased as the absorbed dose increased, with notable differences between the two homopolymers and the blend. The stages of gelation were effectively monitored by means of dynamic rheological measurements, allowing the possible mechanisms of network formation to be elucidated. The doses required for gelation of the PVA, PVP, and blend samples, determined on the basis of the Winter and Chambon criteria for gelation, were found to be 12 kGy for the 12.8 wt% PVA, 4 kGy for the 12.8 wt% PVP, and 5 kGy for the 8 wt% PVA/4.8 wt% PVP solutions. The unexpected lower gelation dose demonstrated by the blend sample, compared with predictions based on the blend composition, and the associated gelation mechanism are also discussed.

Elasticity↗

Tailor-made functional surfaces: potential elastomeric biomaterials I.

In the present investigation, different functional monomers, like hydroxyethyl methacrylate, acrylic acid, N-vinyl pyrrolidone and glycidyl methacrylate, have been grafted onto the surface of EPDM film (approx. 200 microm) using simultaneous photo-grafting (lambda > or = 290 nm) and cold plasma-grafting techniques, to alter the surface properties, such as hydrophilicity and, therefore, biocompatibility. Here, we have carried out simultaneous plasma-grafting, unlike the conventional post plasma-grafting. The effect of different surface grafting techniques on the degree of surface modification and resultant biocompatibility has been investigated. The chemical changes on the polymer backbone are followed from the results of attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS), which shows the peaks corresponding to the functional groups of the monomers grafted onto the film surface. The morphology of the modified surfaces was investigated using scanning electron microscopy (SEM) technique. The induced hydrophilicity and resultant cell compatibility were followed from the water contact angle measurements and in vitro human carcinoma cell adhesion/proliferation tests, respectively. All the grafted samples exhibited variable cell compatibilities depending upon the type of monomer and their degree of grafting; however, always better than the neat samples. Hydroxyethyl methacrylate and acrylic acid showed exceptionally high cell compatibility in terms of cell adhesion and proliferation.

Biocompatible Materials↗

Influence of molecular structure on the synergistic action of theophylline or dipyridamole derivatives in the prostaglandin-type inhibition of platelet aggregation.

Approximately 30 new derivatives of theophylline and dipyridamole have been prepared and examined as potentiators of the inhibition of platelet aggregation induced by the prostaglandin analogue BW 245C. Potentiating activity has been found to be sensitive to molecular size and also to the presence of specific groups. Polymeric adducts based on dextran, poly(ethylene glycol) or poly(N-vinyl pyrrolidone), and aliphatic esters with alkyl chain-lengths greater than 7 are inactive in potentiation. Derivatives containing carboxyl groups are also inactive. Potentiation is discussed in terms of platelet membrane penetration and extra- and intra-cellular processes. The latter are invoked to account for the enhanced potentiation shown by dipyridamole and derivatives when aggregation is induced by PAF-acether rather than ADP. One derivative of particular interest is the adduct of theophylline with 1,2,5,6-diisopropylidene-D-glucose, containing a furanose ring. This is a more active potentiator than theophylline itself, possibly owing to its molecular resemblance to cAMP. On conversion to the pyranose form all activity is removed.

Animals↗