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.     

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.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

Influence of Co2+, Ni2+ and Fe2+ on the production of tetrapyrroles by Methanosarcina barkeri

Summary The selective formation of three tetrapyrroles, Co-containing corrinoids, Ni-containing factor F₄₃₀ and Fe-containing cytochromes (haems) by Methanosarcina barkeri Fusaro (DSM 804) was achieved as a function of the concentrations of Co²⁺, Ni²⁺ and Fe²⁺ in a methanol minimmum medium. It was found that about 70% of the total tetrapyrroles synthesized was excreted into the culture supernatant. Hence, the continuous production of tetrapyrroles in a fixed-bed reactor (supporter: porous diatomaceous clay) was carried out at a dilution rate of 10 day^-1 (850 ml medium/85 ml column/day). The effluent discharged from the reactor contained the excreted tetrapyrroles, the concentrations of which were dependent upon the Co²⁺, Ni²⁺ and Fe²⁺ concentrations in the feed medium. The maximum productivities from the reactor (1 l basis) were 52 M corrinoids/day, 24 M F₄₃₀/day and 8 M haems/day, respectively.     

The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity

Hyaluronidase treatment of hyaluronic acid produced a series of oligosaccharides. Those between 3 and 16 disaccharides in length stimulated angiogenesis in vivo and the proliferation of tissue cultured endothelial cells in vitro. This effect appears to be cell type specific, as no stimulation of fibroblasts or smooth muscle cells was observed. Endothelial cells were found to endocytose both high- and low-molecular-mass hyaluronate, which might be receptor mediated. Fibroblasts and smooth muscle cells, cultured under the same conditions, showed negligible uptake of hyaluronate. Thus, the cell-specific effects may be due to the differences in internalization of hyaluronate. High-molecular-weight hyaluronate both inhibited endothelial cell proliferation and disrupted newly formed monolayers. These data are consistent with the ability of hyaluronate to inhibit new blood vessel formation in vivo and also suggest that hyaluronate metabolism plays a pivotal role in the regulation of angiogenesis.