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 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).     

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.     

The expression of a novel estrogen receptor, GPR30, in epithelial ovarian carcinoma and its correlation with MMP-9

The aim of the present study is to investigate the expression of a novel estrogen receptor, G protein-coupled receptor 30 (GPR30) and its correlation with matrix metalloproteinases-9 (MMP-9) in epithelial ovarian cancer (EOC). Ovary tissues were obtained from 39 female patients, including 30 cases of EOC and 9 cases of benign ovarian tumor. Four normal ovary tissues were used as control. Immunohistochemical staining was used to detect the expressions of GPR30 and MMP-9. Chi square test, Fisher's exact test and Spearman's rank correlation analysis were used for statistical analysis. The results showed that GPR30 overexpression rate in EOC cases was significantly higher than those in benign ovarian tumor and normal ovary cases. Whereas MMP-9 overexpression rate in EOC cases was significantly higher than that in normal ovary cases, without any difference to that in benign ovarian tumor cases. To demonstrate the relationship between GPR30 and clinicopathological variables of EOC, we further analyzed the pathology type, FIGO stage and age of patients sampled in our study. The analysis showed there were significant differences of GPR30 overexpression rate among various pathology types and different FIGO stages (P<0.05), and no significant difference of both GPR30 and MMP-9 among three age groups (P>0.05). Moreover, GPR30 expression was positively correlated with MMP-9 (r(s)=1.000, P=0.002). These results suggest that GPR30 may be involved in the invasion and metastasis of EOC, being a potential index of EOC early diagnosis and malignancy grade prediction.     

Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells(hESCs) can self-renew indefinitely and differentiate into all cell types in the human body.Therefore,they are valuable in regenerative medicine,human developmental biology and drug discovery.A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes.Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin.These hESCs express alkaline phosphatase and hE...     

Chimeras of the integrin beta subunit mid-region reveal regions required for heterodimer formation and for activation.

A central region of the beta2 integrin subunit, RN (residues D300 to C459), was replaced by the equivalent sequences from beta1 and beta7 to give the chimeras beta2RN1 and beta2RN7. Whilst the former construct failed to form heterodimer at the cell surface with alphaL, the later of these could be expressed together with the alphaL subunit to form a variant LFA-1. Based on recent modelling work, the RN region consists of two parts, one is the C-terminal end of the putative A-domain (RB, residues D300 to A359), and the other the mid-region (BN, residues Y360 to C459). Chimeras exchanging the two component regions were made. Of the four resultant chimeras, only the beta2RB1 chimera failed to support LFA-1 expression. Thus the beta1 specific residues of this region affect the interaction with the alphaL subunit. Whereas the alphaL/beta2RB7 LFA-1 variant is wildtype like with respect to ICAM-1 adhesion, the alphaLbeta2BN1 and alphaLbeta2BN7, as well as the alphaLbeta2RN7, variants are more adhesive than the wildtype. These results suggest that an authentic beta2 mid-region is, in part, required for maintaining the LFA-1 in a resting state.     

Chicken IL-6 is a heat-shock gene

The febrile response is elicited by pyrogenic cytokines including IL-6 in response to microorganism infections and diseases in vertebrates. Mammalian HSF1, which senses elevations in temperature, negatively regulates the response by suppressing pyrogenic cytokine expression. We here showed that HSF3, an avian ortholog of mammalian HSF1, directly binds to and activates IL-6 during heat shock in chicken cells. Other components of the febrile response mechanism, such as IL-1β and ATF3, were also differently regulated in mammalian and chicken cells. These results suggest that the febrile response is exacerbated by a feed-forward circuit composed of the HSF3-IL-6 pathway in birds.