Carbohydrate energy metabolism in Fasciola gigantica (trematoda)

Umezurike G. M. and Anya A. O. 1980. Carbohydrate energy metabolism in Fasciola gigantica (Trematoda). International Journal for Parasitology10: 175–180. Adult Fasciola gigantica contained 4.49 ± 0.06 % (mean ± S.D.) wet weight glycogen. Tissue homogenates contained high levels of malate dehydrogenase (MDH), NAD-linked malic enzyme (ME), Phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDH). MDH, PEPCK and ME activities appeared to be localized in both cytosolic and mitoehondrial fractions, fumarase activity appeared to be predominantly mitochondrial whereas LDH and pyruvate kinase activities were cytosolic in distribution. Polyacrylamide gel electrophoresis revealed the predominance of LDH-1, LDH-2 and LDH-3 but only traces of LDH-4 and LDH-5 isoenzymes in the crude cytosolic fraction. LDH activity in the crude sample was inhibited by excess substrate (pyruvate). The mitoehondrial system showed NADH -cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, NADH oxidase and some cytochrome c-oxygen oxidoreductase activities. Under anaerobic conditions, NADH-fumarate oxidoreductase and succinate-NAD + oxidoreductase activities of mitoehondrial preparations were stimulated in the presence of ADP and ATP respectively. Isolated mitochondria contained rhodoquinone and no ubiquinone, and isolated rhodoquinone was readily reduced by succinate in the presence of submitochondrial particles. Hydrogen peroxide was produced by submitochondrial particles in the presence or absence of KCN or in the presence of fumarate.     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

Comparative sensitivities of electrophoretic assays for human enzymes

The sensitivities of 26 starch gel electrophoretic enzyme assays have been compared using HeLa human cells and A9 mouse cells grown in vitro.This research was supported by National Institutes of Health Grant No. USPHS GM 09966.     

Early molecular events in the interaction of enveloped viruses with cells

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells.The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell.Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to a label transfer from virus to cell membranes or vice versa.While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.     

Proteolytic fragmentation of myosin: location of SH-1 and SH-2 thiols.

The heavy chain fragmentation pattern of native myosin when digested by proteolytic enzymes is influenced by such conditions as the nature of the proteolytic agent, ionic strength and presence or absence of divalent cations. HMM and S-1 produced by digestion of 14CNEM-labelled myosin under various conditions were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Purified samples of these species were digested under controlled conditions by chymotrypsin and trypsin and a comparison of the observed heavy chain fragmentation patterns led to a sequential arrangement of the proteolytic fragments. The main features of this arrangement are the following: a 21K molecular weight tryptic peptide is found at the N-terminal side of myosin heavy chain. Adjacent to it is a 48K peptide, then a 19.5K peptide containing the two SH-1 and SH-2 thiols. These three peptides constitute the heavy chain of S-1. Adjacent to this S-1 heavy chain is a tryptic (and also chymotryptic) 40K peptide. The rest of the HMM heavy chain on the C-terminus is a sequence susceptible to both chymotrypsin and trypsin attack yielding an undefined number of small peptides.     

Evolution des activités protéolytiques digestives chez les Jeunes Mulgilidae

Proteolytic activity in the stomach, pyloric caeca and intestine decreases with increasing size in the three fish species Mugil auratus, M. capito and M. saliens. Differences between these species are found mainly in the gastric proteolytic activity which appears to be related to diet. This activity is pronounced in M. saliens whose diet is mainly carnivorous. Comparison of regression lines relating gastric proteolytic activity to size reveals differences which distinguish M. auratus from both of the other species. Total proteolytic activity exhibits high variability depending on the types of diet.     

Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa

Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Mitochondrial abnormalities in human phaeochromocytoma

Summary Mitochondrial abnormalities are reported in four cases of phaeochromocytoma. These abnormalities include swelling and scant cristae, intramitochondrial dense bodies, septate-like junctions, intercristal fusion plus spheroidal bodies, and intramitochondrial rodlets. These structural mitochondrial changes are associated with reduction in activity of the mitochondrial enzymes, monoamine oxidase and succinic dehydrogenase.Supported by a grant-in-aid from the National Health and Medical Research Council