Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.     

Phenylglyoxal modification of the Photosystem II reaction center

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance (ESR), have been used in conjunction with fluorescence-induction and dye-reduction assays to monitor electron transport in Photosystem II (PS II) subchloroplast particles incubated with the covalent modifier, phenylglyoxal. Phenylglyoxal-modified digitonin (D-10) particles from spinach are characterized by a high initial fluorescence yield (Fi) and an abolition of the variable component of fluorescence (Fv); an inhibition of PS-II-mediated reduction of dichlorophenol indophenol (DPIP) by sym-diphenylcarbazide; an abolition of flash-induced absorption transients (t1/2 greater than 2 microseconds) at 820 nm attributed to the primary electron donor, P-680+; the inhibition of photoreduction of the acceptor Qa; and the elimination of the ESR Signal 2s and Signal 2f. These observations suggest the critical participation of specific arginine residues on both the oxidizing and reducing sides of Photosystem II and also implicate phenylglyoxal as a quinone-binding site inhibitor (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271).     

Observation on a 65-kilodalton protein isolated from kanagawa positive strains of Vibrio parahaemolyticus

Crude hemolysin from four KP+ strains of Vibrio parahaemolyticus belonging to serotype 02:K3 exhibited a major protein band (molecular weight, 65 kilodaltons (kDa] in addition to a previously known thermostable direct hemolysin band (molecular weight, 21 kDa) in SDS - polyacrylamide slab gel electrophoresis. These strains showed maximum virulence leading to 100% mouse lethality within 2-6 h. It is hypothesized that this 65-kDa protein may play a vital role in the pathogenesis of the disease caused by V. parahaemolyticus.     

Methylamine does not inhibit rates of endogenous lipolysis in isolated myocardial cells from rat heart

Triacylglycerol lipase activity with a pH optimum of 5 was present in homogenates of myocardial cells from rat heart. Acid lipase activity was inhibited by serum, heparin, and increased ionic strength. Methylamine, a lysosomotropic agent, did not inhibit the basal or isoproterenol-stimulated rate of endogenous lipolysis as measured by glycerol output from control myocytes. Similarly, accelerated rates of glycerol output that are a consequence of an elevation in the intracellular stores of triacylglycerols in myocytes from diabetic rat hearts and from myocytes prepared with free fatty acids in the isolation solutions were not reduced by methylamine. Therefore, the acid lysosomal triacylglycerol lipase must not be involved in the mobilization of endogenous triacylglycerols in myocardial cells from rat heart.     

Gibberellic acid by solid state fermentation: Consistent and improved yields

Five strains each of Gibberella fujikuroi and Fusarium monoliforme were screened to select G. fujikuroi P-3, a strain capable of giving consistent production of gibberellic acid (GA(3)) by solid state fermentation (SSF). The comparative production of GA(3) by SSF and submerged fermentation (SmF) indicated better productivity with the former technique. The accumulation of GA(3) was 1.626 times higher in the case of SSF. On the basis of available carbohydrates in the media, the percent conversions were 0.096 and 0.156 in SmF and SSF, respectively. The use of coarse wheat bran of the particle size of 0.3-0.4 cm resulted in an increase of 2.5 times in the yield of GA(3). The enrichment of commercial wheat bran with soluble starch gave enhanced accumulation to an extent of 3.5 times. The relation between GA(3) production and cell growth in SSF was similar to that encountered in SmF. The consistent and improved yields to a tune of 1.22 g GA(3) per kilogram dry moldy bran (DMB) establish the potential and feasibility of SSF for the production of GA(3) by G. fujikuroi P-3. On preliminary cost analysis, a net savings of about 60% and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SmF technique was evident.     

Effect of phosphate supplementation on oxygen delivery at high altitude

In the present communication, effect of low doses of phosphate supplementation on short-term high altitude adaptation has been examined. Studies were carried out in 36 healthy, male, sea-level residents divided in a double blind fashion into drug and placebo treated groups. 3.2 mmol of phosphate were given orally to each subject of the drug treated group once a day for 4 days on arrival at an altitude of 3,500 m. Sequential studies were done in the subjects in both groups on the 3rd, 7th, 14th and 21st day of their altitude stay. Haemoglobin, haematocrit, erythrocyte and reticulocyte counts increased to the similar extent in both groups. Blood pH, pO₂ and adenosine tri-phosphate (ATP) did not differ between the two groups. On 3rd day of the altitude stay, inorganic phosphate and 2,3-diphosphoglycerate (2,3 DPG) levels in the drug treated group increased significantly as compared to the placebo group. No significant difference in inorganic phosphate and 2,3 DPG was observed later on in the two groups. Psychological and clinical tests also indicated that the drug treated subjects felt better as compared to the placebo treated subjects. The present study suggests that low doses of phosphate increases circulating 2,3-DPG concentration which in turn brings about beneficial effect towards short term high altitude adaptation.     

Presence of nucleosomes inPenicillium chrysogenum

We have studied the chromatin structure ofPenicillium chrysogenum. This fungus presents the typical nucleosomal repeat and the core DNA size characteristic of all the eukaryotes. The repeat length (about 180 base pairs) is in the range of those obtained for most fungi (160–180 base pairs) and shorter than in higher eukaryotes. Knowledge aboutP. chrysogenum chromatin structure opens the way to the study of the mechanisms of genetic regulation in this filamentous fungus.     

Aging and cerebral aminopeptidase activity

A comparative study of brain aminopeptidase activity between 18 month old male rats and young adults of 3 months has been carried out utilizing the arylamides Leu-, Arg-, Lys-, Tyr- and Asp-beta-naphthylamide as substrates. Statistical analysis of results showed no significant differences either in areas studied or for enzymatic activities detected when both ages were compared. Two different patterns of regional distribution of enzymatic activity were observed: One came to be as a result of the use of Lys-, Arg-, Leu- or Tyr-beta-naphthylamide and the other as a result of the use of Asp-beta-naphthylamide.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Skinned muscle fibers of Aplysia: potentiation of contraction by "background" calcium and lack of effect of cyclic AMP

1. Aplysia buccal muscle E1 can be skinned with saponin in a low ionic strength medium. Pulses of calcium, which were ineffective at causing contraction in intact fibers, elicited contraction in skinned fibers. 2. Tension in skinned fibers increased at [Ca2+] greater than 10(-7) M and was maximal at 6 x 10(-7) M. 10(-5) M [Ca2+] caused irreversible damage to the fibers. 3. Fibers did not exhibit "catch", i.e. they relaxed quickly upon removal of calcium. 4. Optimal pH for tension was 7.0. 5. Contractile responses to calcium pulses were increased by raising "background" [Ca2+] to 10(-7) M. 6. Cyclic AMP (10(-4) and 10(-3) M) had no effect on tension.