Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

The chemical identity of the immunoreactive LHRH-like peptide biosynthesized in the human placenta

Freshly obtained human placental trophoblasts were minced and pulselabeled for 30 min at 37°C with tritiated L-Tyrosine. After homogenisation, the crude extract was centrifuged and deproteinized with 10% TCA. The supernatant was defatted and the peptides concentrated through hydrophobic binding on ODS-silica cartridges. The bound, crude peptide extract was eluted and subjected to gradient, reverse-phase High Performance Liquid Chromatography. The fractions corresponding to the absorption peak of reference, synthetic LHRH were collected and extensively purified to radioactive homogeneity by further multiple HPLC. After digestion with pyroglutamate aminopeptidase, the resulting nonapeptide was manually sequenced by dansyl-Edman degradation. All the incorporated radioactivity was found to reside exclusively in residue number 4 of the nonapeptide; thus establishing for the first time the primary sequence of biosynthetic placental LHRH as: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, identical to its hypothalamic counterpart.     

The phosphorylation of brain microtubular proteins in situ and in vitro

The phosphorylation of microtubular proteins isolated by reassembly in vitro from slices of guinea-pig cerebral cortex labelled with [32P]orthophosphate was investigated. Under the conditions tested, both and the alpha and beta forms of tubulin contained metabolically-active P which accounted for about one third of the total 32P incorporated into protein; the remaining protein-bound 32P was associated with 3-4 minor high MW components co-purifying with tubulin during two cycles of assembly-disassembly. Microtubular protein prepared in this way contained approx. 0.8 mol of alkalilabile P/mol of tubulin dimer (M.W. 110,000). In vitro studies showed that reassembled microtubular protein preparations catalysed the incorporation of up to 0.55 mol of P/mol of tubulin dimer during incubation with Mg2+ and [gamma 32P]ATP. The reaction was linear during the first 30 min of incubation at 37 degrees C. Cyclic AMP (10 microM, final concentration) caused a transient increase in the initial rates of tubulin phosphorylation. Little label was incorporated into the minor high M.W. components under these conditions. The in vitro phosphorylation of microtubular protein increased in a non-linear manner with respect to protein concentration: this was in contrast to earlier experiments showing linear kinetics when chromatographically isolated tubulin was tested for intrinsic kinase activity. Isolated microtubular protein preparations bound [3H]GTP, [3H]ATP and to a lesser extent, [3H]cyclic AMP, and exhibited Ca(2+)-ATPase activity (up to 60 pmol Pi released min/mg protein at 37 degrees C).     

A Human Active Lower Limb Model for Chinese Pedestrian Safety Evaluation

A subsystem impactor test for pedestrian lower limb injury evaluation has been brought in China New Car Assessment Protocol(CNCAP).Concerning large anthropometr...     

Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation,Growth and Fusion

Lipid droplets （LDs） were once viewed as simple, inert lipid micelles. However, they are now known to be organelles with a rich proteome involved in a myriad of cellular processes. LDs are heterogeneous in nature with different sizes and compositions of phospholipids, neutral lipids and proteins. This review takes a focused look at the roles of proteins involved in the regulation of LD formation, expansion, and morphology. The related proteins are summarized such as the fat-specific protein （Fsp27）, fat storage-inducing trans- membrane （FIT） proteins, seipin and ADP-ribosylation factor 1-coat protein complex I （Arf-COPI）. Finally, we present important challenges in LD biology for a deeper understanding of this dynamic organelle to be achieved.     

长时程增强诱导和维持过程中海马CA1区神经细胞粘附分子蛋白水平与mRNA表达的变化

既往研究发现,神经细胞粘附分子(neural cell adhesion molecules,NCAM)对海马CA1区突触传递长时程增强(longterm potentiation,LTP)的诱导和维持极为关键。本文采用原位杂交法和Western blot法,观察了大鼠海马腑片LTP诱导和维持过程中NCAM mRNA和蛋白水平的动态变化过程。结果显示,强直刺激诱发fEPSP斜率升高10 min时,海马CA1区NCAM mRNA染色阳性神经元数量显著增加(76.6±11.5个),NCAM蛋白水平亦明显升高(7.190±0.64任意单位/50μg蛋白)。强直刺激诱发fEPSP斜率升高1 h时,NCAM mRNA染色阳性神经元数量为73.3±14.0个,NCAM蛋白量为9.031±0.71任意单位/50 μg蛋白;与强直刺激后10 min比较,NCAM mRNA表达无显著变化,而NCAM蛋白水平变化明显。NMDA受体特异阻断剂AP-5在损害LTP的同时,显著抑制NCAM mRNA和蛋白的增加。实验结果表明,在大鼠海马LTP诱导和维持过程中,NCAM mRNA增强的表达相对稳定,而NCAM蛋白水平呈现先低后高的变化。