Role of DNA barcoding in marine biodiversity assessment and conservation: An update

More than two third area of our planet is covered by oceans and assessment of marine biodiversity is a challenging task. With the increasing global population, there is a tendency to exploit marine resources for food, energy and other requirements. This puts pressure on the fragile marine environment and necessitates sustainable conservation efforts. Marine species identification using traditional taxonomical methods is often burdened with taxonomic controversies. Here we discuss the comparatively new concept of DNA barcoding and its significance in marine perspective. This molecular technique can be useful in the assessment of cryptic species which is widespread in marine environment and linking the different life cycle stages to the adult which is difficult to accomplish in the marine ecosystem. Other advantages of DNA barcoding include authentication and safety assessment of seafood, wildlife forensics, conservation genetics and detection of invasive alien species (IAS). Global DNA barcoding efforts in the marine habitat include MarBOL, CeDAMar, CMarZ, SHARK-BOL, etc. An overview on DNA barcoding of different marine groups ranging from the microbes to mammals is revealed. In conjugation with newer and faster techniques like high-throughput sequencing, DNA barcoding can serve as an effective modern tool in marine biodiversity assessment and conservation.     

Inhibition of glucose 6-phosphatase by pure and impure C-type phospholipases. Reactivation by phospholipid dispersions and protection by serum albumin.

1. Pure or impure C-type phospholipases hydrolysed rat liver microsomal phosphatides in situ at 5 degrees or 37 degrees C. At 5 degrees C mean hydrolysis of total phospholipids was 90% by Bacillus cereus and 75% by Clostridium perfringens (Clostridium welchii) C-type phospholipases. 2. Four degrees of inhibition of glucose 6-phosphatase (D-glucose 6-phosphate phosphohydrolase; EC 3.1.3.9) resulted. (a) At 37 degrees C inhibition was virtually complete and apparently irreversible. (b) At 5 degrees C phospholipase C inhibited 50-87% of the activity expressed by intact control microsomal fractions. (c) Bovine serum albumin present during delipidation alleviated most of this inhibition: at 5 degrees C phospholipase C plus bovine serum albumin inhibited by 0-35% (mean 18%):simultaneous stimulation by the destruction of its latency seems to offset glucose 6-phosphatase inhibition, sometimes completely. (d) If latency was first destroyed, phospholipase C plus bovine serum albumin inhibited 30-50% of total glucose 6-phosphatase activity at 5 degrees C. Only this inhibition is likely largely to reflect the lower availability of phospholipids, essential for maximal enzyme activity, as it is virtually completely reversed by added phospholipid dispersions. Co-dispersions of phosphatidylserine plus phosphatidylcholine (1:1, w/w) were especially effective but Triton X-100 was unable effectively to restore activity. 3. Considerable glucose 6-phosphatase activity survived 240min of treatment with phospholipase C at 5 degrees C, but in the absence of substrate or at physiological glucose 6-phosphate concentrations the delipidated enzyme was completely inactivated within 10min at 37 degrees C. However, 80mM-glucose 6-phosphate stabilized it and phospholipid dispersions substantially restored thermal stability. 4. It is concluded that glucose 6-phosphatase is at least partly phospholipid-dependent, and complete dependence is not excluded. For reasons discussed it is impossible yet to be certain which phospholipid class(es) the enzyme requires for activity.     

Design and synthesis of glucagon partial agonists and antagonists

The hyperglycemia and ketosis of diabetes mellitus are generally associated with elevated levels of glucagon in the blood. This suggests that glucagon is a contributing factor in the metabolic abnormalities of diabetes mellitus. A glucagon-receptor antagonist might provide important evidence for glucagons's role in this disease. In this work we describe how we combined structural modifications that led to glucagon analogues with partial agonist activity to give glucagon analogues that can act as competitive antagonists of glucagon-stimulated adenylate cyclase activity. Using solid-phase synthesis methodology and preparative reverse-phase high-performance liquid chromatography, we synthesized the following seven glucagon analogues and obtained them in high purity: [D-Phe4,Tyr5,Arg12]glucagon (2); [D-Phe4,Tyr5,Lys17,18]glucagon (3); [Phe1,Glu3,Lys17,18]glucagon (4); [Glu3,Val5,Lys17,18]glucagon (5); [Asp3,D-Phe4,Ser5,Lys17,18]glucagon (6); I4-[Asp3,D-Phe4,Ser5,Lys17,18]glucagon (7); [Pro3]glucagon (8). Purity was assessed by enzymatic total hydrolysis, by chymotryptic peptide mapping, and by reverse-phase high-performance liquid chromatography. The new analogues were tested for specific binding, for their effect on the adenylate cyclase activity in rat liver membranes, and for their effect on the blood glucose levels in normal rats relative to glucagon. Analogues showing no adenylate cyclase activity were examined for their ability to act as antagonists by displacing glucagon-stimulated adenylate cyclase dose-response curves to the right (higher concentrations). The binding potencies of the new analogues relative to glucagon (= 100) were respectively 1.0 (2), 1.3 (3), 3.8 (4), 0.4 (5), 1.3 (6), 5.3 (7), and 3 (8). Glucagon analogues 3-5 and 8 were all weak partial agonists with EC50 values of 500 (3), 250 (4), 1600 (5), and 395 nM (8), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.     

Why choline supplementation did not enhance phosphatidylcholine level in Candida albicans

In contrast to Saccharomyces cerevisiae cells, the supplementation of the growth media of Candida albicans cells with choline did not result in PC enrichment. The level of accumulation of choline uptake, which is the first step of its utilisation was found to be 50% higher in S. cerevisiae cells. However, the activity of choline kinase (EC 2.7.1.32), the first enzyme in CDP-choline pathway was identical between the two cell types. It appears that CTP: phosphocholine cytidylyl-transferase (EC 2.7.7.15) may be the regulatory enzymatic step in overall PC biosynthesis of C. albicans cells.     

Inhibitory effects of methylxanthines on the activity of xanthine oxidase

The $\text{in vitro}$ and $\text{in vivo}$ effects of three methylxanthines caffeine, theophylline and theobromine on the activity of the enzyme xanthine oxidase (EC 1.2.3.2.) was investigated with a view to understand their biochemical action. The studies revealed all the three methylxanthines to be inhibitors of the milk xanthine oxidase activity and the inhibition was found to be competitive in nature. The preincubation studies indicated a greater inhibition of the enzyme with the methylxanthines. Excessive amount of the substrate (2.5 × 10^?4M) resulted in progressive inhibition of the enzyme activity. Low concentrations of methylxanthines exerted a definite inhibitory effect on the xanthine oxidase activity at lower substrate concentrations. At higher concentrations of the substrate, the inhibitory effect due to the same concentration of methylxanthines did not produce any added inhibition of the enzyme activity to that produced by the substrate alone. However, added inhibition by high concentrations of methylxanthines was detectable even when the enzyme activity was markedly inhibited by higher concentrations of the substrate. The $\text{in vivo}$ administration of methylxanthines caused a significant inhibition of the xanthine oxidase activity in lungs, kidneys, heart and brain of rats. Consequently, the level of uric acid in the tissues of the drug treated animals was also found to be reduced.     

Interactions of glucagon and glucagon analogs with isolated canine hepatocytes

We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.     

Effect of phosphatidylserine enrichment on amino acid transport in yeast

A 1.5- to 3.5-fold accumulation of phosphatidylserine was observed when Candida albicans and Saccharomyces cerevisiae cells were grown in the presence of hydroxylamine, a known inhibitor of phosphatidylserine decarboxylase. However, as compared to S. cerevisiae cells, the levels of phosphatidylcholine and phosphatidylethanolamine were much lower in C. albicans cells. The enrichment of phosphatidylserine selectively affected the transport of several amino acids.