Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

Pycnogenol® and vitamin E inhibit ethanol‐induced apoptosis in rat cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol‐induced activation of caspase‐3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 261–271, 2004     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

谷子和狗尾草核型分析

作者研究了我国谷子(Setaria italica Beauv.)和狗尾草的核型与Giemsa带,两者核型基本相同,均为2n=18=14m+2sm+2st(SAT)。带型亦相近,另外在谷子和狗尾草中都发现有四倍体。     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.