An efficient, practical synthesis of 2-methoxyestradiol

An efficient and practical scheme to synthesize 2-methoxyestradiol has been developed. The key step was the copper-mediated methoxylation using ethyl acetate as a co-catalyst to introduce a methoxyl group. These synthetic procedures of four steps from 17β-estradiol as starting material gave 2-methoxyestradiol with a 61% overall yield.     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.     

A quantitive evaluation of gap junctions and their morphological alteration during differentiation of amphibian notochord cells

Summary The early development of notochord cells may be divided into three phases according to the quantitative evaluation of gap junctions: late gastrula, neurula and from tailbud to tadpole. In late gastrula, the percentage of the area of gap junctions to total membrane is 0.054% and most of the gap junctions are small in size. During the stages of neurulation, the ratios of gap junctions to total membrane area increase and remain high (0.106–0.181 %), and the majority of the gap junctions are of medium and large size. The high ratios of gap junctions to membrane area during neurulation suggests that intercellular communication via gap junctions is important during this period. In the stages from tailbud to tadpole the ratios decrease and drop drastically to 0.001 % and most of the gap junctions found are small in size. It is in the last phase that gap junctions of altered configuration appear.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.     

Molecular cloning,expression patterns and subcellular localization of porcine <Emphasis Type="Italic">TMCO1</Emphasis> gene

The product of transmembrane and coiled-coil domains 1 (TMCO1) gene is a member of DUF841 superfamily of several eukaryotic proteins with unknown function. The partial DNA sequence of porcine TMCO1 was first cloned with a pig 567 bp ORF encoding 188 amino acids. By tissues expression analysis, the TMCO1 was found highly expressed in the liver, kidney and heart. The porcine TMCO1 protein was subsequently demonstrated to localize in the mitochondrion by confocal fluorescence microscopy. This data provides an important basis for conducing further studies on the functions and regulatory mechanisms underlying the role of TMCO1 gene.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles.

Intravenous administration of APC such as splenocytes loaded with a soluble protein Ag has been shown to prime for an Ag-specific CTL response. It is thought that the APC directly presents loaded Ag in a MHC-restricted manner. However, it is demonstrated in this study that allogeneic splenocytes, MHC-free RBC, and even synthetic lipid vesicles (liposomes) after loading with OVA can elicit an OVA-specific and MHC-restricted CTL response. Biodistribution studies of these Ag-associated vehicles showed that the liver, spleen, and lung were the major organs responsible to scavenge these carriers, suggesting that the monocyte-macrophage system was involved in the Ag presentation for CTL. Depletion of macrophages by a specific macrophage killer, Cl2MDP, containing liposomes, abolished the CTL induction by immunization with OVA Ag carried by these vehicles except the induction by syngeneic splenocytes. Thus, the syngeneic splenocytes present Ag directly to the T cells, but other membranous vehicles carry the Ag to the host APC including macrophages, which then present it to the T cells. These results indicate that formulation of an Ag in membranous/colloidal vehicles may be a way to prime for a CTL response.