Use of cultured human fibroblasts for rapid evaluation of cytotoxic effects of bioactive substances

A test system for detecting cytotoxic effects of bioactive substances based on human fibroblast culture is proposed. The effects of acrylamide, streptomycin, cycloheximide, sodium dodecyl sulfate, sanguiritrine, and ethanol were evaluated by organic stain binding. Typical dose-effect relationships were detected for all substances except cycloheximide. The proposed test system can be used for screening of bioactive substances in preclinical trials. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 5, pp. 587–590, May, 2000     

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake

Summary The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a K_M of about 2 × 10^–4 M for both media and a V of about 5 nmol min^–1mg^–1 and 2 nmol min^–1mg^–1 for derepressed and repressed cells respectively. The pho 1–118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1–118 mutation.     

Hymenolepis diminuta (order cyclophyllidea): Histochemical localization of glycogen,neutral lipid,and alkaline phosphatase in developing worms

Summary The distribution and concentration of glycogen, neutral lipid and alkaline phosphatase were studied histochemically in developmental stages of the tapeworm, Hymenolepis diminuta. Examination of immature, mature and gravid proglottids from worms of selected ages showed a general trend of increasing concentration of glycogen, neutral lipid and alkaline phosphatase from the anterior to the posterior of the worm. However, no significant difference was observed in the concentration of these substances in individual proglottid types from worms of various ages.A portion of a thesis submitted by LFM to the Graduate School of the University of Nevada, Reno, in partial fulfillment of the requirements for the Master of Science in Biology.     

Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase

Summary A Yarrowia lipolytica gene library was constructed in vector YRp7 and transformed into a Saccharomyces cerevisiae strain lacking both major acid phosphatase activities. A 2.18 kb genomic sequence restoring the ability to hydrolyze -naphthyl phosphate was isolated. Its sequencing revealed an ORF encoding 358 amino acids without significant homology with any known phosphatase. A putative signal peptide and several possible sites for N-glycosylation were identified. Phosphate-regulated expression of the cloned gene was observed in Y. lipolytica. Disruption data favoured the hypothesis that it might encode a minor phosphatase species.     

Two genes control three alkaline phosphatases in Schizosaccharomyces pombe

Summary Mutants of Schizosaccharomyces pombe defective in alkaline phosphatase activity have been selected and analyzed. The mutations map in two different loci, pho2 and pho3, both located on the right arm of chromosome II. Pho2-mutants lack the activity of a soluble alkaline phosphatase specific for nitrophenyl-phosphate. Its activity requires Mg ions and it is abolished with Zn ions. The pho3-mutations block the activities of a soluble and of a membrane-bound nonspecific alkaline phosphatase. Both pho3-controlled enzyme forms exhibit similar substrate specificities, pH-optima and Mg ions are essential for both activities. Zn ions can partially replace the Mg ions. The three enzyme forms migrate differently on nondenaturing polyacrylamide gels. We speculate that pho2 and pho3 represent the structural genes for a substrate-specific and two forms of a nonspecific alkaline phosphatase.     

Effect of lidocaine on cholera vibrio-induced changes in the jejunum, renal medulla, and lungs of suckling rabbits

Lidocaine injected into suckling rabbits infected with a virulent strain ofVibrio cholerae abolishes the development of hydropic and ballooning degeneration the jejunal enterocytes. Secretory granules and lipid inclusions accumulate in jejunal enterochromaffin cells and interstitial cells of renal medulla, respectively, and are not released into the vascular bed. In pulmonary tissue ultrastructural changes are mild, and capillary epithelium is undamaged, indicating that lidocaine stimulates pulmonary enzymes which inactivate biologically active substances implicated in the pathogenesis of cholera. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 6, pp. 632–637, June, 1997     

On-line monitoring of solutes in dialysate using wavelength-dependent absorption of ultraviolet radiation

The aim of the study was to assess the wavelength dependence of the UV absorbance during monitoring of different compounds in the dialysate. UV absorbance was determined by using a double-beam spectrophotometer on dialysate samples taken at pre-determined times during dialysis, over a wavelength range of 180–380 nm. Concentrations of several removed substances, such as urea, creatinine, uric acid, phosphate andβ ₂-microglobulin, were determined in the blood and in the spent dialysate samples using standard laboratory techniques. Millimolar extinction coefficients, for urea, creatinine, monosodium phosphate and uric acid were determined during laboratory bench experiments. The correlation between UV absorbance and substances both in the dialysate and in the blood was calculated at all wavelengths. A time-dependent UV absorbance was determined on the collected dialysate samples from a single dialysis session over a wavelength range of 200–330 nm. The highest contribution from observed compounds relative to the mean value of the absorbance was found around 300 nm and was approximately 70%. The main contribution to the total absorbance from uric acid was confirmed at this wavelength. The highest correlation for uric acid, creatinine and urea was obtained at wavelengths from 280 nm to 320 nm, both in the spent dialysate and in the blood. The wavelength region with the highest correlation for phosphate andβ ₂-microglobulin, with a suitable UV-absorbance dynamic range, was from 300 to 330 nm. In the wavelength range of 220–270 nm the highest absorbance sensitivity for the observed substances was obtained. A suitable wavelength range for instrumental design seems tobe around 290–330 nm. The relatively high correlation between UV absorbance and the substances in the spent dialysate and in the blood indicates that the UV-absorbance technique can estimate the removal of several retained solutes known to accumulate in dialysis patients.     

The Effect of haemolymph extraction on distribution of lysosomal enzymes in Lymnaea stagnalis haemocytes: A cytochemical study

An enzyme cytochemical, light microscopy study was undertaken to evaluate the effect of haemolymph extraction on distribution of acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase in haemocytes of Lymnaea stagnalis. The study revealed that discrete acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase-staining subpopulations of haemocytes do exist in L. stagnalis, and that there are differences in distribution. A high percentage, about 96%, of haemocytes showed positive reaction for peroxidase, the percentage of haemocytes showing activity for alpha-naphthyl acetate esterase, acid phosphatase, and alkaline phosphatase, was about 54, 42, and 34, respectively. Haemolymph extraction by foot retraction, significantly affected enzyme distribution. Whereas distribution of acid phosphatase increased significantly from day 1 to 5 after extraction of haemolymph, that of all other enzymes showed significant decrease from day 1 to 7 depending upon the enzyme. The distribution of all enzymes stabilized by day 8 after extraction of haemolymph. Probable reasons for the observed fluctuations in the distribution of enzymes subsequent to haemolymph extraction are discussed.     

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

Taste and pheromone perception in the fruit fly Drosophila melanogaster

Taste is an essential sense for detection of nutrient-rich food and avoidance of toxic substances. The Drosophila melanogaster gustatory system provides an excellent model to study taste perception and taste-elicited behaviors. “The fly” is unique in the animal kingdom with regard to available experimental tools, which include a wide repertoire of molecular-genetic analyses (i.e., efficient production of transgenics and gene knockouts), elegant behavioral assays, and the possibility to conduct electrophysiological investigations. In addition, fruit flies, like humans, recognize sugars as a food source, but avoid bitter tasting substances that are often toxic to insects and mammals alike. This paper will present recent research progress in the field of taste and contact pheromone perception in the fruit fly. First, we shall describe the anatomical properties of the Drosophila gustatory system and survey the family of taste receptors to provide an appropriate background. We shall then review taste and pheromone perception mainly from a molecular genetic perspective that includes behavioral, electrophysiological and imaging analyses of wild type flies and flies with genetically manipulated taste cells. Finally, we shall provide an outlook of taste research in this elegant model system for the next few years.     

The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe

Summary The pho4 gene of the fission yeast Schizosaccharomyces pombe is regulated by thiamin. The nucleotide sequence of this gene is given here and it is shown that it matches the amino acid sequence of thiamin-repressible acid phosphatase, corroborating genetic evidence that pho4 represents the structural gene of this enzyme. The gene codes for a protein of 463 amino acids in length and shows regions of strong similarity with the phosphate-repressible acid phosphatase of Schizosaccharomyces pombe. The enzyme has a cleavable signal sequence 18 amino acids long and carries nine potential N-glycosylation sites.     

Thiamine in Schizosaccharomyces pombe: dephosphorylation,intracellular pool,biosynthesis and transport

Summary We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellulary mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.     

Analgesic effects of spinal cord peptide factors produced during the establishment and development of generators of pathologically enhanced excitation in the rat spinal cord

In rats with a pain syndrome caused by the production of a generator of pathologically enhanced excitation, substances of peptide nature with analgesic properties were found to be produced in the nociceptive system of the spinal cord. Spinal cord extracts derived from rats with such syndromes (pain syndrome of spinal origin or adjuvant arthritis) exerted analgesic effects when injected intraventricularly into recipient rats with a pain syndrome of spinal origin. The highest analgesic activity was displayed by extracts obtained from the region where the generator of pathologically enhanced excitation had been set up. The analgesic activity of the extracts was directly related to the severity and duration of the pain syndrome in the donor rats. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N^no 10, pp. 374–377, October, 1994     

Hyicin 3682, a bioactive peptide produced by Staphylococcus hyicus 3682 with potential applications for food preservation

Bacteriocins are peptides produced by bacteria and having inhibitory activity against other bacteria. Many of these substances may be useful as antibacterial agents for practical applications. In this study, 21 Staphylococcus spp. isolated from pigs, dogs and bovine milk in different states of Brazil were investigated for staphylococcin production. Hyicin 3682, a bacteriocin produced by one such strain, inhibited almost all strains tested, including Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. PCR experiments showed that hyicin 3682 is lantibiotic-related, but not identical, to both epidermin and Bsa. The maximum production of hyicin 3682 (6,400 AU/ml) was observed after 24 h of growth in BHI medium at 37 °C. Hyicin 3682 proved to be a cationic, small antimicrobial peptide with a molecular mass of 2,139 Da. It exhibited resistance to low pH and to heating at 65 °C, and partial sensitivity to proteolytic enzymes. Taken together, these results suggest that hyicin 3682, the first bacteriocin characterized in Staphylococcus hyicus, has potential biotechnological applications as a food preservative. Moreover, hyicin 3682 was able to inhibit its producer strain, suggesting that an effective immune system for specific protection against hyicin 3682 is not found in its producer strain, a characteristic not described thus far for other staphylococcins.     

Isoforms of alkaline phosphatase from mouse internal organs after bilateral adrenalectomy

The activity of alkaline phosphatase of female CBA and BALB/c mice is studied after bilateral adrenalectomy. Interstrain differences in enzyme activity are revealed in some organs of the control and experimental animals. The expression of new isoforms of alkaline phosphatase in hypocorticoidism is demonstrated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3 pp. 262–264, March, 1996 Presented by V. P. Kaznacheev, Member of the Russian Academy of Medical Sciences     

Radiometric evaluation of the effect of xenobiotics on proliferation of bone marrow cells in mice

A radiometric method of evaluating the effect of chemical compounds on the proliferative activity of bone marrow cells is described which consists of measuring the incorporation of labeled³H-thymidine in DNA. Results are reported on a comparative study of the effect of known immunotropic substances on bone marrow cell proliferation using the present method and the method of evaluating endogenous colony formation. Analysis of the results obtained by two variants ofin vivo andin vitro experiments provides additional information regarding the mechanism of action of the substances. Translated fromByulleten Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 222–224, August, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Morphocytochemical characteristics of cerebellar neuronal populations in fishes with various ambulatory activities

The cytoplasmic distribution of basophilic substance and the number of chromatin granules in nuclei of cerebellar neurons were studied. Neuronal proteins were assayed in the molecular, ganglionic, and granular cerebellar layers in fishes of various ecological and morphological groups. A quantitative analysis of Nissl bodies and chromatin granules revealed a polymorphism of neuronal populations. Protein concentrations per body volume of stellate and Purkinje cells in pelagic fishes were higher than in benthophages by 7.9% and 12.3%, respectively. However, the content of protein substances in granular cells of pelagic fishes was 8.9% lower than in those cells of benthophages. Tinctorial heterogeneity of cell populations and peculiarities of the distribution of protein substances in various neurons reflect specific features of structural and functional organization of the cerebellum in fishes with different ambulatory activities. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 7, pp. 81–84, July, 1999     

Alterations in growth, haematopoiesis and serum chemistry profiles infitness 14226SB mutant mice

We investigated patterns of growth, haematopoiesis and alterations in serum chemistry profiles that were associated with anN-ethyl-N-nitrosourea-induced mutation in thefitness 1 locus in chromosome 7 in mice. Mice hemizygous for thefit 1 mutation [c fit 1 ^4226SB /Df(c Mod2 sh1) ^VT ] had growth retardation characterised by decreased body weights, shorter body lengths and tail lengths at 40 and 60 days of age compared to normal homozygous control mice [c ^ch +/ c ^ch +]. Haemograms revealed that mice hemizygous for thefit 1 ^226SB mutation that were killed at 40 days had microcytic, hypochromic anaemia that was mildly regenerative in nature. No significant differences were detected in total leucocyte counts, differential leucocyte counts and platelet counts between hemizygous mutant mice and control mice. Haemograms from the surviving mice hemizygous for thefit 1 ^4226SB mutation that were killed at 60 days indicated that the haemoglobin concentrations, mean corpuscular volumes and mean corpuscular haemoglobin concentrations were significantly decreased compared to normal controls. The reticulocyte counts and the platelet counts from the mice hemizygous for thefit 1 ^4226SB mutation were mildly increased compared to normal controls killed at 60 days. Serum chemistry analyses at 40 days of age indicated that the hemizygousfit 1 mutant mice had hypoproteinaemia characterised by hypoalbuminaemia and hypoglobulinaemia. They also developed hypoglycaemia, mild hyperphosphataemia and moderate elevation in the activity of alkaline phosphatase in serum. Quantitative isozyme analysis indicated that the increase in total serum alkaline phosphatase activity in the hemizygous mutant mice was due to an increase in the bone isozyme. In addition to those alterations in the serum chemistry profile noted above, hemizygous mutant mice killed at 60 days had an extremely mild increase in urea nitrogen concentration and mildly increased activities of alanine aminotransferase and aspartate aminotransferase in serum. From these data, it was concluded that thefit 1 ^4226sb mutation m mice causes growth retardation, microcytic, hypochromic anaemia, and alterations in serum chemistry profiles that suggest lesions in one or more organ system(s). The exact mechanism(s) by which thefit 1 ^4226SB mutation mediates these lesions remains to be elucidated.     

Evaluation of biological activity of toxic agents in a unicellular model

Combined toxicity of inorganic substances: Ag(I)−Cu(II), Cr(VI)−Ni(II) is studied in a culture ofSaccharomyces cerevisiae yeasts. The dynamics of yeast growth in the presence of individual metals an their combinations is examined. A diagram method for the evaluation of combined toxicity is proposed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 594–600, May, 1997