Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

The relation between the variability of polygenic morphological and monogenic biochemical traits as exemplified by Karakul sheep

A quantitative method for investigation of relationship between polygenic and monogenic traits has been proposed. It is based on examination of relationship between frequencies of distribution classes of an adaptive quantitative trait and frequencies of certain genetic character in the same classes. The method permits to locate a gene marker within a space of quantitative trait values. Using adaptively significant morpho-anatomic traits, it is possible to estimate indirectly adaptive values of gene markers under consideration, since, in accordance with the concept of adaptive norm, "average" phenotypes have maximal fitness, whereas deviative phenotypes transgress the bounds of the optimum. As a genetical character, genotype of certain biochemical locus, individual heterozygosity range or interlocus combinations of alleles could be used. The method has been applied to newborn Astrakhan lambs. Principal component analysis has been used to obtain complex characterization for six constitutional characters. Some regularities in location of homo- and heterozygous genotypes of the transferrin locus within a space of morphological characters' values have been revealed.     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Identification and characterization of a major early cytomegalovirus DNA-binding protein

We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.     

Parasitism byEphedrus cerasicola [Hym.: Aphidiidae] developing in different stages ofMyzus persicae [Hom.: Aphididae]

The parasitoidEphedrus cerasicola Stary oviposited in the 4 nymphal instars and in newly moulted apterous adults ofMyzus persicae (Sulzer). Development and reproduction of unparasitized and parasitized aphids at 21°C were compared. Unparasitized aphids developed to adults in 6.5 days and started to reproduce after 7 days. Longevity varied between 7 and 42 days. Net reproductive rate (R₀) was 40.7. In contrast to older nymphs, aphids parasitized in the 1 st instar almost never reached the adult stage before mummification. Aphids parasitized in 2nd, 3rd and 4th instar and as newly moulted adults produced respectively 0.07 %, 2 %, 23 % and 32 % of offspring produced by unparasitized aphids. Corresponding reproductive periods were 1, 1.4, 3 and 4 days. Host age at parasitization had a slight effect on the parasitoid's developmental rate and had no effect on egg or pupal survival, or on the sex ratio of the emerging parasitoids.