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PubMed1992

Selective purification of two distinct protein kinases (C-kinase and casein kinase II) from the membrane fraction of mouse brain by NED-affinity column chromatography.

By means of NED-affinity column chromatography, two distinct protein kinases have been selectively purified from the crude membrane extract of mouse brain. One (designated P-I kinase) was eluted from the column by the buffer containing 5 mM EGTA and the other (designated P-II kinase) was eluted by the buffer containing 0.6 M KCl. The activity of A-kinase was detected in the column passed through fraction. Biochemical characteristics of P-I and P-II kinases corresponded exactly to those of C-kinase and casein kinase II (CK-II), respectively. In addition, immunoprecipitate experiment using anti-CK-II antiserum against the beta-subunit of Drosophila CK-II showed that P-II kinase is identical to CK-II and the 62 kDa cellular polypeptide is associated with the kinase.

Animals↗

PubMed2006

[Construction of pMSCV recombinant retroviral vector containing polo-like kinase 3(plk3) cDNA and its effects on cell proliferation].

AIM: To construct pMSCV retroviral vector (RV) containing Polo-like kinase 3(plk3) cDNA, a new member of Ser/Thr protein kinase family, and to study the function of plk3 gene by introducing it into human K562 and HL60 cells. METHODS: plk3 cDNA was sub-cloned into retroviral vector pMSCV-puroR to generate pMSCV-plk3-puroR, pMSCV-puroR being used as the control. RVs were generated by introducing RV vectors into 293T-Amphotropic packaging cells. Titers of RV were measured by NIH3T3 cells with Large-scale-real-time titration method (LaSRT). K562 and HL60 cells were infected with pMSCV-plk3-puroR or pMSCV-puroR viral supernatant by flow-through transduction method, respectively. Following 3-day selection with 3 mg/L puromycin, K562-plk3-puroR, HL60-plk3-puroR, K562-puroR and HL60-puroR cells were obtained. PCR was used to identify the integration of plk3 gene in K562-plk3-puroR and HL60-plk3-puroR cells. Transduction rates were determined by clone-forming ability against puromycin in semi-solid culture medium. Cell growth curve was measured by MTT colorimetry, the cell cycle and apoptotic analysis were detected by flow cytometry, etc. RESULTS: Virus titers generated was (1.31+/-0.65)x10(9)TU/L. The K562 and HL60 transfection efficiency reached 82.3%+/-3.70% and 62.9%+/-4.94%, respectively (n=3). After 3 d puromycin selection, purified K562-plk3-puroR and HL60-plk3-puroR cell were obtained. The proliferation of K562 and HL60 cells was slowed down by plk3 gene transduction. Compared with wild type K562 cells: (1) when cultured in complete DMEM medium, more K562-plk3-puroR cells were observed in G(0)-G(1), 49.7%+/-3.38% vs 43.9%+/-2.34% (P=0.03); (2) when cultured in serum-free DMEM medium for 10 h, fewer K562-plk3-puroR cells were observed in S phase, 43.6%+/-3.74% vs 54.5%+/-1.52% (P=0.02) with lower apoptosis rate, 3.4%+/-0.37% vs 1.3%+/-0.31% (P=0.01). CONCLUSION: Introduction of plk3 gene into tumor cells with RV vectors can slow down cell proliferation, prevent cells into cell cycle and protect cells from apoptosis in serum-free culture.

Animals↗

PubMed1991

Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2006

River capture, range expansion, and cladogenesis: the genetic signature of freshwater vicariance.

River capture is potentially a key geomorphological driver of range expansion and cladogenesis in freshwater-limited taxa. While previous studies of freshwater fish, in particular, have indicated strong relationships between historical river connections and phylogeographic pattern, their analyses have been restricted to single taxa and geological hypotheses were typically constructed a posteriori. Here we assess the broader significance of river capture among taxa by testing multiple species for the genetic signature of a recent river capture event in New Zealand. During the Quaternary an upper tributary of the Clarence River system was diverted into the headwaters of the Wairau River catchment. Mitochondrial DNA (control region and cytochrome b) sequencing of two native galaxiid fishes (Galaxias vulgaris and Galaxias divergens) supports headwater exchange: populations from the Clarence and Wairau Rivers are closely related sister-groups, whereas samples from the geographically intermediate Awatere River are genetically divergent. The upland bully Gobiomorphus breviceps (Eleotridae), in contrast, lacks a genetic signature of the capture event. We hypothesize that there is an increased likelihood of observing genetic signatures from river capture events when they facilitate range expansion, as is inferred for the two galaxiid taxa studied here. When river capture merely translocates genetic lineages among established populations, by contrast, we suggest that the genetic signature of capture is less likely to be retained, as might be inferred for G. breviceps. Rates of molecular evolution calibrated against this recent event were elevated relative to traditional estimates, consistent with the contribution of polymorphisms to branch lengths at shallow phylogenetic levels prior to fixation by purifying selection and drift.

Animals↗

PubMed1989

Development and clinical application of DNA probe specific for Peptostreptococcus micros.

P. micros is thought to be an important pathogen in the etiology of certain inflammatory lesions, however, the role of this microorganism is uncertain due to the lack of rapid and reliable method to identify this species. The purpose of this study was to develop a DNA probe specific for the detection of P. micros in order to evaluate its prevalence in oral infectious lesions. The whole genomic DNA from P. micros was partially digested and inserted into the vector pUC 13. Four recombinant clones were selected, purified and screened against reference strains of Peptostreptococcus species to check the species specificity and then applied to clinical isolates. The sensitivity and specificity of the DNA probe for P. micros was 99.2% and 100%, respectively. P. micros could be detected in 7.2% of subgingival dental plaques from the patients with adult periodontitis and in 16.1% of the endodontic lesions with periapical pathosis. In the endodontic lesions, there was a good correlation between the clinical symptoms and the presence of P. micros. These data strongly suggested that the DNA probe can be useful in the detection of P. micros and that this microorganism is important in certain periodontal and endodontic lesions.

Aggressive Periodontitis↗

PubMed1985

An amino acid sequence common to both cartilage proteoglycan and link protein.

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. In the course of determining the primary structure of link protein, two peptides produced by digestion of rat chondrosarcoma link protein with trypsin or chymotrypsin have been selectively purified by immunoaffinity chromatography on a column of monoclonal anti-link protein antibody (8A4) immobilized to Sepharose 4B. These peptides have been sequenced using the double-coupling dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate procedure. A consensus sequence, Cys-X-Ala-Gly-Trp-Leu-X-Asp-Gly-Ser-Val-X-Tyr-Pro-Ile-X-X-Pro, obtained by comparing the affinity-isolated tryptic peptide with the affinity-isolated chymotryptic peptide and an overlapping tryptic peptide, shows homology with a sequence obtained from the NH2-terminal of a CNBr peptide from proteo glycan core protein of bovine nasal cartilage: Ser-Ser-Ala-Gly-Trp-Leu-Ala-Asp-Arg-Ser-Val-Arg-Tyr-Pro-Ile-Ser-. We suggest that the common sequence is structurally important to the function of these proteins and may be involved in the binding of both link protein and proteoglycan to hyaluronic acid.

Amino Acid Sequence↗

PubMed2026

Complete mitochondrial genomes and phylogenetic analysis of three species of Indo-Pacific freshwater gobies, Stiphodon (Gobiiformes,Oxudercidae).

Stiphodon is a genus of gobioid fishes found primarily in freshwater streams on islands throughout the Indo-Pacific region. It is the most speciose genus of Sicydiinae, with more than three dozen named species. Complete mitochondrial genome (mtDNA) sequences were determined for three species, namely Stapledon atropurpureus, Stapledon elegans, and Stapledon semoni. Maximum likelihood and Bayes inference inferences from 13 protein-coding genes (11,433 bp) or the COXI gene (1,551 bp) alone were made on these three together with four mitogenomes previously available. S. percnopterygionus and S. tuivi formed an outgroup to the other five Stiphodon species, meanwhile Stapledon atropurpureus and S. semoni were recovered as sister species. The ratios of the non-synonymous to the synonymous ubstitution rates for all PCGs were in the range 0\ (Ka/Ks) \1, which suggests they have been subject to purifying selection.

Animals↗

PubMed1983

Fine resolution of the poly ADP-ribosylated domains of polynucleosomal chromatin: DNA gene and integrity analysis; mechanism of histone H1 modification.

The focus of our laboratory has been to ascertain how the poly(ADP-ribosyl)ation reaction influences the structure and biological function of nucleosomal chromatin. Antibody to poly(ADP-ribose (ADP-Rib] was coupled to Sepharose to prepare an immunoaffinity column. The following new information concerning poly(ADP-Rib) and chromatin was obtained with this column: 1) those limiting domains of nucleosomal chromatin undergoing the modification (circa 10%) could be isolated in the "bound" fraction, 2) antibody-bound nucleosomes contained all the poly ADP-ribosylated acceptors and polymerase activity of the bulk chromatin, 3) bound nucleosomes contain significant numbers of internal DNA strand breaks, and [3H]-thymidine (TdR) repair incorporation from in vivo "DNA repair label," compared to the unbound, bulk of the chromatin, 4) the presence of actively transcribed genes in "bound" nucleosomes is being investigated, 5) the acetylation modification of histones occurs in the same domains of chromatin as does poly(ADP-ribosyl)ation, and 6) poly ADP-ribosylated histone H1 can be selectively purified by the immunoaffinity method. These same histone H1 molecules appear to be equally accessible to the histone kinase, phosphorylation modification. In addition, new information has been obtained concerning histone H1 cross-linking by poly(ADP-ribosyl)ation, and on polymerase binding sites to chromatin. We have reconstituted histone H1-depleted chromatin with intact H1 and peptide domains of H1, and subsequently studied H1-poly(ADP-Rib)-complex synthesis. The data indicate that elongation of poly(ADP-Rib) proceeds on the amino terminal region of this histone. By utilizing the new techniques of DNA technology, huge advances in our understanding of the programmed structure of the eukaryotic genome have been accomplished in a relatively short time. To complement this explosion of information, it is important to ascertain how these recently appreciated properties of eukaryotic DNA are packaged within extended and condensed domains of chromatin. The research to be discussed is directed, in part, at this latter topic. We have initiated a program aimed at furthering our comprehension of chromatin structure by studying one specific, enzymatically active chromosomal protein.

Chemical Phenomena↗

PubMed1983

[The thymopause: an immunological climacteric].

The part played by the thymus and its products in conferring and maintaining immunological competence is examined. Attention is also devoted to the effect of thymic involution on the immune system, with particular reference to a fall in circulating thymic hormone. Generally speaking, this fall occurs in the 5th decade of life, which can be described as the thymopause. It is accompanied by increased sensitivity to infection and neoplasia. Two lines of intervention are suggested: the use of highly purified, selective hormone fractions after careful evaluation of the functional aspects of the immune system; employment of organ extracts containing all or nearly all the thymic hormones and peptides in an integrated form. It is felt that the second approach is more practicable today, since it permits broad-spectrum interventions through the stimulation of many mechanisms with different sequential steps, and a well-programmed biological rhythm over the course of time.

Aged↗

PubMed1993

In vitro candidastatic properties of the human neutrophil calprotectin complex.

Microbiostatic mechanisms may contribute substantially to host defense against infection by certain microbes. We studied the candidastatic activity of human neutrophils, neutrophil cytosol, and neutrophil-derived "calprotectin," a cytosolic protein complex comprised of two subunits, MRP8 and MRP14. Intact neutrophils, neutrophil lysates (prepared by ultrasonic disruption, freezing and thawing, or nonionic detergent extraction), and granule-depleted neutrophil cytosol were effective in restricting the growth of Candida albicans in a nutrient-rich tissue culture medium, RPMI 1640. Neither a subcellular fraction enriched in neutrophil granules nor selected purified granule components (lactoferrin, myeloperoxidase, cathepsin G, leukocyte elastase, lysozyme, and defensins) exerted candidastatic activity in this medium. Gel filtration liquid chromatography, anion exchange FPLC, and SDS-PAGE showed that the fungistatic factor in neutrophil cytosol was associated with the calprotectin complex. Its antifungal effects included restriction of yeast phase and mycelial growth and inhibition of glucose incorporation by yeast phase cultures. The antifungal effects of calprotectin were sustained for over 120 h and were inhibited by zinc. However, studies with 65Zn-enriched RPMI suggested that the candidastatic effects of calprotectin were not mediated by sequestration or binding of zinc. After reversed phase HPLC, calprotectin fractions containing MRP14 exhibited fungistatic activity, whereas fractions depleted of MRP14 but enriched for MRP8 lacked fungistatic activity. The results support a potentially significant role for the calprotectin complex of neutrophil cytosol in antifungal defense and suggest that MRP14 is of key importance in that activity.

Antifungal Agents↗

PubMed1995

Noninvasive detection of putative biomarkers for colon cancer using fecal messenger RNA.

Deaths from colon cancer number over 60,000 each year in the United States. Because early detection results in a high cure rate, development of noninvasive techniques for detection of colon cancer has received much interest. The ability to detect early changes in colonocyte genes and gene expression would provide valuable information. We have shown previously that alterations in protein kinase C (PKC) isoform expression are associated with changes in colonic cell proliferation, a key intermediate marker for the prediction of tumorigenesis. Here, we describe a method for the quantitative detection of mRNAs for select PKC isoforms isolated from rat feces containing exfoliated colonocytes. After total RNA extraction from fresh fecal material, polyadenylated RNA was selectively purified and quantitated with slot blotting and hybridization to oligodeoxythymidylic acid. Fecal polyadenylated RNA was used for semiquantitative (mimic) RT-PCR to quantitate PKC isoform mRNA expression. We detected mRNA for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-sigma, but not for PKC-beta or PKC-gamma, which is consistent with the profile of isoforms detected previously in scraped colonic mucosa using immunoblot analysis. This noninvasive method, utilizing feces containing exfoliated colonocytes, is a sensitive noninvasive technique for quantitating luminal mRNAs. This provides a means to monitor gene expression of colonic epithelial cells, which may have predictive value in monitoring the neoplastic process.

Animals↗

PubMed1996

A rapid method for purification of monoclonal human IgM from mass culture.

We describe in this paper the rapid and cheap purification of human immunoglobulin M from hybridoma supernatant of mass culture. The method consists of two steps: 1. concentration of supernatant by ultrafiltration and 2. dialysis against distilled water, pH 6.4. To produce the supernatant, the hybridomas are grown in RPMI media with fetal calf serum (10% FCS) in 250 ml flasks under normal tissue culture conditions. More than 14 mg IgM can be nearly selectively purified within 6 h from 5 l of antibody containing hybridoma supernatant. Most of the IgM molecules stay in a penta- and monomeric form and are positive in physiological and immunohistochemical studies.

Antibodies, Monoclonal↗

PubMed1998

The MHC-E locus is the most well conserved of all known primate class I histocompatibility genes.

The HLA-E locus is characterized by limited polymorphism and low levels of cell surface expression. However, the function of the products of this nonclassical MHC class I gene remains unknown. To evaluate the conservation of the MHC-E locus throughout anthropoid primate evolution, we identified the homologue of the HLA-E locus in six different New World monkey species. Full-length sequencing of MHC-EcDNAs in four unrelated cotton-top tamarins (Saguinus oedipus) revealed no evidence for polymorphism. Using the PCR, denaturing gradient gel electrophoresis, and direct sequencing, we also identified MHC-E alleles in five other New World monkey species, representing all extant platyrrhine families. In contrast to all other classical and nonclassical MHC class I genes in primates, the rate of synonymous nucleotide substitution is much greater than the rate of nonsynonymous nucleotide substitution within exons 2 and 3 encoding the peptide binding region (PBR) in MHC-E genes. The PBR of the MHC-E molecule, therefore, has evolved under purifying selective pressures, and the very unusual evolutionary history of this ancient gene provides further evidence that the products of the HLA-E locus serve a critical immunological function. Given the remarkable conservation of the PBR during primate evolution, this critical immunological function is probably related to the peptide binding ability of the MHC-E protein.

Amino Acid Sequence↗

PubMed2002

Selection in the evolution of gene duplications.

BACKGROUND: Gene duplications have a major role in the evolution of new biological functions. Theoretical studies often assume that a duplication per se is selectively neutral and that, following a duplication, one of the gene copies is freed from purifying (stabilizing) selection, which creates the potential for evolution of a new function. RESULTS: In search of systematic evidence of accelerated evolution after duplication, we used data from 26 bacterial, six archaeal, and seven eukaryotic genomes to compare the mode and strength of selection acting on recently duplicated genes (paralogs) and on similarly diverged, unduplicated orthologous genes in different species. We find that the ratio of nonsynonymous to synonymous substitutions (Kn/Ks) in most paralogous pairs is <<1 and that paralogs typically evolve at similar rates, without significant asymmetry, indicating that both paralogs produced by a duplication are subject to purifying selection. This selection is, however, substantially weaker than the purifying selection affecting unduplicated orthologs that have diverged to the same extent as the analyzed paralogs. Most of the recently duplicated genes appear to be involved in various forms of environmental response; in particular, many of them encode membrane and secreted proteins. CONCLUSIONS: The results of this analysis indicate that recently duplicated paralogs evolve faster than orthologs with the same level of divergence and similar functions, but apparently do not experience a phase of neutral evolution. We hypothesize that gene duplications that persist in an evolving lineage are beneficial from the time of their origin, due primarily to a protein dosage effect in response to variable environmental conditions; duplications are likely to give rise to new functions at a later phase of their evolution once a higher level of divergence is reached.

Animals↗

PubMed2005

Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.

Aptamers, Nucleotide↗

PubMed1989

The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified Gi, measured with GTPase and phospholipase C assays.

We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (Kd = 0.34 nM) and low affinity (Kd = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low Km GTPase activity. Kyotorphin produced a stimulation of low Km GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low Km GTPase. The kyotorphin stimulation of low Km GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, Gi, but not with Go. Similar evidence of selective coupling of kyotorphin receptor to Gi was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with Gi but not with Go. These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through Gi. This study provides the first evidence of a selective involvement of Gi in the receptor-mediated activation of phospholipase C.

Animals↗

PubMed1990

Antibody to a soluble protein purified from brain selectively labels layer V corticofugal projection neurons in rat neocortex.

An antibody to a soluble protein (protein 36) isolated and purified from rat brain labels the cell bodies and processes of pyramidal cells within layer V of the rat neocortex. We have used the fluorescent retrograde axonal tracer, Fast blue, in combination with FITC immunocytochemistry to determine the projection sites of the cortical neurons detected by this antibody. Retrogradely labeled pyramidal tract neurons and corticotectal neurons are labeled with the protein 36 antibody, but the callosally projecting neurons within layer V are not. Thus within the neocortex the antibody to protein 36 may selectively detect a particular class of neuron, the corticofugal projection neurons of layer V.

Animals↗

PubMed2004

Selective inhibition of purified human phosphodiesterase 4A expressed in yeast cell GL62 by ciclamilast, piclamilast, and rolipram.

AIM: To improve the specific activity of human phosphodiesterase 4A (PDE4A) expressed in yeast cell GL62 and investigate the effects of selective phosphodiesterase 4 (PDE4) inhibitors (ciclamilast, piclamilast, and rolipram), selective phosphodiesterase 5 (PDE5) inhibitor zaprinast, and cyclooxygenase (COX) inhibitors (aspirin, indomethacin) on human PDE4A activity expressed in yeast cell GL62. METHODS: Human PDE4A was expressed in yeast cell GL62 after CuSO4 induction and the specific activity of human PDE4A was improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. The activity of PDE4A was measured by high performance liquid chromatography (HPLC). RESULTS: Induced PDE4A activity expressed in crude yeast cell GL62 supernatant and pellet was (340+/-21) nmol/g/min and (250+/-25) nmol/g/min respectively. The specific activity of recombinant PDE4A in supernatant was improved 6.4 fold. Ciclamilast, piclamilast, and rolipram could inhibit PDE4A activity. The IC50 values (95 % confidence limits) of ciclamilast, piclamilast, and rolipram were 1.27 (0.84-1.91), 66.4 (33.3-132.2), and 3.73 (2.51-5.53) micromol/L respectively. Zaprinast, aspirin, and indomethacin had no obvious inhibitory effect on PDE4A activity. CONCLUSION: The specific activity of PDE4A expressed in yeast cell GL62 can be improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. Ciclamilast, piclamilast, and rolipram can inhibit PDE4A activity while zaprinast, aspirin, and indomethacin have no obvious inhibitory effect on PDE4A activity. Human PDE4A expressed in GL62 might be useful in the research and screening of new selective PDE4 inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗