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Biomedical subjects

R Shen

Publications and source records attributed to R Shen.

At least 55 records · Page 3Linked to original sources
PubMed1999

Regulation of mRNA export by nutritional status in fission yeast.

We have isolated a mutation in nup184(nup184-1) that is synthetically lethal with the mRNA export defective rae1-167 mutation in Schizosaccharomyces pombe. The consequence of the synthetic lethality is a defect in mRNA export. The predicted Nup184p is similar to Nup188p of Saccharomyces cerevisiae, and a Nup184p-GFP fusion localizes to the nuclear periphery in a punctate pattern. The Deltanup184 null mutant is viable and also is synthetically lethal with rae1-167. In a rae1(+) background, both the nup184-1 and Deltanup184 mutations confer sensitivity to growth in nutrient-rich medium (YES) that is accompanied by nuclear poly(A)+ RNA accumulation. Removal of the cAMP-dependent protein kinase, Pka1p, relieved the growth and mRNA export defects of nup184 mutants when grown in nutrient-rich medium. The activation of Pka1p is necessary, but not sufficient, to cause the severe poly(A)+ RNA export defects when nup184 mutant cells are incubated in YES, suggesting nutritional status can also regulate poly(A)+ RNA export. Our results suggest that the regulation of poly(A)+ RNA export by Pka1p kinase appears to be indirect, via a translation-dependent step, but post-translationally in response to YES.

Cell Nucleus↗
PubMed1999

Effects of qigong on late-stage complex regional pain syndrome.

CONTEXT: Despite the growing popularity of qigong in the West, few well-controlled studies using a sham master to assess the clinical efficacy of qigong have been conducted. OBJECTIVE: To study the effect of qigong on treatment-resistant patients with late-stage complex regional pain syndrome type I. DESIGN: Block-random placebo-controlled clinical trial. SETTING: Pain Management Center at New Jersey Medical School. PATIENTS: 26 adult patients (aged 18 to 65 years) with complex regional pain syndrome type I. INTERVENTIONS: The experimental group received qi emission and qigong instruction (including home exercise) by a qigong master. The control group received a similar set of instructions by a sham master. The experimental protocol included 6 forty-minute qigong sessions over 3 weeks, with reevaluation at 6 and 10 weeks. Assessment included comprehensive medical history, physical exam, psychological evaluation, necessary diagnostic testing. Symptom Check List 90, and the Carleton University Responsiveness to Suggestion Scale. MAIN OUTCOME MEASURES: Thermography, swelling, discoloration, muscle wasting, range of motion, pain intensity rating, medication usage, behavior assessment (activity level and domestic disability), frequency of pain awakening, mood assessment, and anxiety assessment. RESULTS: 22 subjects completed the protocol. Among the genuine qigong group, 82% reported less pain by the end of the first training session compared to 45% of control patients. By the last training session, 91% of qigong patients reported analgesia compared to 36% of control patients. Anxiety was reduced in both groups over time, but the reduction was significantly greater in the experimental group than in the control group. CONCLUSIONS: Using a credible placebo to control for nonspecific treatment effects, qigong training was found to result in transient pain reduction and long-term anxiety reduction. The positive findings were not related to preexperimental differences between groups in hypnotizability. Future studies of qigong should control for possible confounding influences and perhaps use clinical disorders more responsive to psychological intervention.

Adolescent↗
PubMed1998

Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting.

Myostatin, a member of the transforming growth factor-beta superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene's expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Adult↗
PubMed1998

Additional possible tools for identification of proteins on one- or two-dimensional electrophoresis.

Additional, essentially chemical, identification methods of proteins in polyacrylamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C-side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90 degrees C for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioacetate vapor at 50 degrees C for 6-24 h. The products were analyzed by mass spectrometry-peptide mass fingerprinting. A new type of C-terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90 degrees C for 2-16 h provided cleavages at the C-side of aspartic acid and the N-side of serine/threonine and simultaneous successive truncation at the C-termini of the cleaved fragments. The product resulted in C-terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at -5 degrees C for 8 h deblocked the N-formyl group, and the vapor at 20 degrees C for 4 h deblocked pyrrolidone carboxylate. N-acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50 degrees C for 1 h, followed by reaction with p-sulfophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C-terminal sequencing of protein from polyacrylamide gels is also described.

Amino Acid Sequence↗
PubMed1998

A randomised placebo controlled trial of suppressive acyclovir in late pregnancy in women with recurrent genital herpes infection.

OBJECTIVE: To evaluate the efficacy and safety of a suppressive course of acyclovir in late pregnancy in women with recurrent genital herpes infection on the incidence of viral shedding, herpes lesion development and caesarean section for recurrent genital herpes. DESIGN: Double-blind, randomised placebo controlled clinical trial. SETTING: A department of genitourinary medicine in Sheffield and an antenatal clinic in London. POPULATION: Pregnant women with recurrent genital herpes infection at < 36 weeks of gestation. METHODS: Participating women were given acyclovir 200 mg four times a day (or matching placebo) from 36 weeks of gestation until the time of delivery. Women were seen weekly and viral cultures were obtained from the cervix and vulva. Decisions regarding mode of delivery were left to the discretion of the attending obstetrician. MAIN OUTCOME MEASURES: Delivery by caesarean section for recurrent genital herpes infection. Number of episodes of recurrent genital herpes infection and number of episodes of asymptomatic viral shedding during the treatment period. In addition blood was taken at two weekly intervals to determine acyclovir levels. RESULTS: The total number of women recruited was 63 (31 received acyclovir and 32 received placebo). The number of women undergoing delivery by caesarean section for recurrent herpes at the time of delivery was 12 (19%). The odds ratio for delivery by caesarean section in women taking acyclovir, compared with those taking placebo, was 0.44 (95% CI 0.09-1.59). The odds ratio for clinical recurrences during treatment was 0.10 (95% confidence interval 0.00-0.86) and the odds ratio for clinical recurrence or asymptomatic shedding during treatment was 0.32 (95% CI 0.05-1.56). CONCLUSION: This trial was unable to demonstrate that acyclovir can significantly decrease the number of caesarean section deliveries; however, the number of clinical recurrences was significantly reduced. Two episodes of asymptomatic virus shedding both occurred in women taking acyclovir. At the present time there is little evidence to suggest that acyclovir should be used outside randomised controlled trials for the suppression of recurrent genital herpes infection during pregnancy.

Acyclovir↗
PubMed1998

The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women.

Measurements of total and free testosterone levels in women have lacked precision and accuracy because of limited assay sensitivity. The paucity of normative data on total and free testosterone levels in healthy women has confounded interpretation of androgen levels in women with human immunodeficiency virus (HIV) infection and other disease states. Therefore, the objectives of this study were to develop sensitive assays for the measurement of the low total and free testosterone levels in women to define the range for these hormones during the normal menstrual cycle and assess the total and free testosterone levels in HIV-infected women. By using a larger volume of serum, increasing the incubation time, and reducing the antibody concentration, the sensitivity of the total testosterone assay was increased to 0.008 nmol/L, and that of the free testosterone assay was increased to 2 pmol/L. The mean percent free testosterone was 1.0 +/- 0.1% of the total testosterone. Serum total and free testosterone levels in the follicular and luteal phases were not significantly different, but both demonstrated a modest preovulatory increase, 3 days before the LH peak. Serum total [0.50 +/- 0.32 (14.60 +/- 9.22) vs. 1.2 +/- 0.7 nmol/L (34.3 +/- 21.0 ng/dL); P < 0.0001] and free testosterone levels (5.56 +/- 2.70 (1.58 +/- 0.80) vs. 12.8 +/- 5.5 pmol/L (3.4 +/- 1.7 pg/mL); P < 0.0001) were significantly lower in HIV-infected women (n = 37) than in healthy women (n = 34). Serum total and free testosterone levels were also significantly lower in HIV-infected women who were menstruating normally. There were no significant differences in serum total and free testosterone levels between those who had lost weight and those who had not. Testosterone levels correlated inversely with plasma HIV ribonucleic acid copy number. Serum FSH, but not LH, levels were significantly higher in HIV-infected women than in controls. Using assays with sufficient sensitivity, we defined the range for total and free testosterone levels during the normal menstrual cycle. Serum total and free testosterone levels are lower in HIV-infected women and correlate inversely with plasma HIV ribonucleic acid levels. The hypothesis that androgen deficiency contributes to wasting in HIV-infected women remains to be tested.

Adult↗
PubMed1998

Effects of testosterone replacement with a nongenital, transdermal system, Androderm, in human immunodeficiency virus-infected men with low testosterone levels.

Although weight loss associated with human immunodeficiency virus (HIV) infection is multifactorial in its pathogenesis, it has been speculated that hypogonadism, a common occurrence in HIV disease, contributes to depletion of lean tissue and muscle dysfunction. We, therefore, examined the effects of testosterone replacement by means of Androderm, a permeation-enhanced, nongenital transdermal system, on lean body mass, body weight, muscle strength, health-related quality of life, and HIV-disease markers. We randomly assigned 41 HIV-infected, ambulatory men, 18-60 yr of age, with serum testosterone levels below 400 ng/dL, to 1 of 2 treatment groups: group I, two placebo patches (n = 21); or group II, two testosterone patches designed to release 5 mg testosterone over 24 h. Eighteen men in the placebo group and 14 men in the testosterone group completed the 12-week treatment. Serum total and free testosterone and dihydrotestosterone levels increased, and LH and FSH levels decreased in the testosterone-treated, but not in the placebo-treated, men. Lean body mass and fat-free mass, measured by dual energy x-ray absorptiometry, increased significantly in men receiving testosterone patches [change in lean body mass, +1.345 +/- 0.533 kg (P = 0.02 compared to no change); change in fat-free mass, +1.364 +/- 0.525 kg (P = 0.02 compared to no change)], but did not change in the placebo group [change in lean body mass, 0.189 +/- 0.470 kg (P = NS compared to no change); change in fat-free mass, 0.186 +/- 0.470 kg (P = NS compared to no change)]. However, there was no significant difference between the 2 treatment groups in the change in lean body mass. The change in lean body mass during treatment was moderately correlated with the increment in serum testosterone levels (r = 0.41; P = 0.02). The testosterone-treated men experienced a greater decrease in fat mass than those receiving placebo patches (P = 0.04). There was no significant change in body weight in either treatment group. Changes in overall quality of life scores did not correlate with testosterone treatment; however, in the subcategory of role limitation due to emotional problems, the men in the testosterone group improved an average of 43 points of a 0-100 possible score, whereas those in the placebo group did not change. Red cell count increased in the testosterone group (change in red cell count, +0.1 +/- 0.1 10(12)/L) but decreased in the placebo group (change in red cell count, -0.2 +/- 0.1 10(12)/L). CD4+ and CD8+ T cell counts and plasma HIV copy number did not significantly change during treatment. Serum prostate-specific antigen and plasma lipid levels did not change in either treatment group. Testosterone replacement in HIV-infected men with low testosterone levels is safe and is associated with a 1.35-kg gain in lean body mass, a significantly greater reduction in fat mass than that achieved with placebo treatment, an increased red cell count, and an improvement in role limitation due to emotional problems. Further studies are needed to assess whether testosterone supplementation can produce clinically meaningful changes in muscle function and disease outcome in HIV-infected men.

Absorptiometry, Photon↗
PubMed1998

[The biodegradation of trichloroethylene by a methanotrophic bacterium].

A Methylomonas (strain GYJ3) isolated in our laboratory was identified as the type II methanotroph on the basis of the intractytoplasmic membrane of the ultrastructure. The optimal culture conditions for production of the soluble from of methane monooxygenase (MMO) were determined, in which the ratio of methane to air in atmosphere was 2 to 1 and Cu2+ concentration was 1.5 mumol/L. The biodegradation of trichoroethylene(TCE) by the resting cells of the strain GYJ3 was studied. All experiments were performed with cells grown under above conditions and thus expressing soluble MMO. This results showed that TCE at the high concentration of 30 mg/L did not inhibit to the enzymes in the cells. Addition of formate increased the initial specific TCE degradation rates. The product of TCE oxidation was found to be toxic to the cells. The degree of inactivation of MMO was proportional to the amount of TCE degraded. The TCE degradation capacities(Tc) of resting cells was determined. In no-formate and formate-fed experiments, the TCE degradation capacities were found to be 0.0778 and 0.0851 mg of TCE/mg of dry cell, respectively.

Biodegradation, Environmental↗
PubMed1997

Direct evidence for a soluble methane monooxygenase from type I methanotrophic bacteria: purification and properties of a soluble methane monooxygenase from Methylomonas sp. GYJ3.

The hydroxylase and reductase components of a soluble methane monooxygenase from type I methanotrophs--Methylomonas sp. GYJ3--were purified by a multiple-step LC procedure. The hydroxylase (approximately 240 kDa, determined by an HPLC-size exclusion chromatography method) has three subunits with molecular masses of 56, 43, and 27 kDa, suggesting that the enzyme has an (alphabeta gamma)2 subunit structure. The HPLC method was developed to purify the hydroxylase component, and the purified protein has a specific activity of 541 nmol propene oxide x mg(-1) protein x min(-1), which is two times the specific activity of the protein purified by the two-step LC procedure. The iron content in the hydroxylase purified by the two-step LC procedure is 2.1 mol of Fe per mole of protein, but the iron content in the protein by the HPLC procedure is 3.78 mol of Fe per mole of protein. The diversity of iron contents in this protein is due mainly to the use of different purification methods. The reductase has a molecular mass of 42 kDa. The UV-VIS spectrum of the protein is similar to that of proteins from other methanotrophs, suggesting that the protein contains a FAD cofactor and a [2Fe-2S] center. The partially purified component B stimulated the MMO activity of the hydroxylase and reductase system by 40-fold.

Chromatography, High Pressure Liquid↗
PubMed1997

Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2.

Shp-1, Shp-2 and corkscrew comprise a small family of cytoplasmic tyrosine phosphatases that possess two tandem SH2 domains. To investigate the biological functions of Shp-2, a targeted mutation has been introduced into the murine Shp-2 gene, which results in an internal deletion of residues 46-110 in the N-terminal SH2 domain. Shp-2 is required for embryonic development, as mice homozygous for the mutant allele die in utero at mid-gestation. The Shp-2 mutant embryos fail to gastrulate properly as evidenced by defects in the node, notochord and posterior elongation. Biochemical analysis of mutant cells indicates that Shp-2 can function as either a positive or negative regulator of MAP kinase activation, depending on the specific receptor pathway stimulated. In particular, Shp-2 is required for full and sustained activation of the MAP kinase pathway following stimulation with fibroblast growth factor (FGF), raising the possibility that the phenotype of Shp-2 mutant embryos results from a defect in FGF-receptor signalling. Thus, Shp-2 modulates tyrosine kinase signalling in vivo and is crucial for gastrulation during mammalian development.

Animals↗
PubMed1997

Molecular characterization of specific interactions between SHP-2 phosphatase and JAK tyrosine kinases.

Interactions between SHP-2 phosphotyrosine phosphatase and JAK tyrosine kinases have recently been implicated in cytokine signal transduction. However, the molecular basis of these interactions is not well understood. In this study, we demonstrate that SHP-2 is tyrosine-phosphorylated by and associated with JAK1 and JAK2 but not JAK3 in COS-1 cell cotransfection experiments. SHP-2 phosphatase activity appears not to be required for JAK and SHP-2 interactions because SHP-2 with a mutation at amino acid 463 from Cys to Ser, which renders SHP-2 inactive, can still bind JAKs. We further demonstrate that SHP-2 SH2 domains (amino acids 1-209) are not essential for the association of JAKs with SHP-2, and the region between amino acids 232 and 272 in SHP-2 is important for the interactions. Furthermore, tyrosine residues 304 and 327 in SHP-2 are phosphorylated by JAKs, and phosphorylated SHP-2 can associate with the downstream adapter protein Grb2. Finally, deletion of the N terminus but not the kinase-like domain of JAK2 abolishes the association of JAK2 with SHP-2. Taken together, these studies identified novel sequences for SHP-2 and JAK interactions that suggest unique signaling mechanisms mediated by these two molecules.

Animals↗
PubMed1997

Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export.

To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167. Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa). This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae. Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery. In contrast to NIC96, the npp106 gene is not essential. Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus. A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions. In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106. We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality. These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.

Amino Acid Sequence↗
PubMed1997

A deletion mutation in the SH2-N domain of Shp-2 severely suppresses hematopoietic cell development.

Shp-1 and Shp-2 are cytoplasmic protein tyrosine phosphatases that contain two Src homology 2 (SH2) domains. A negative regulatory role of Shp-1 in hematopoiesis has been strongly implicated by the phenotype of motheaten mice with a mutation in the Shp-1 locus, which is characterized by leukocyte hypersensitivity, deregulated mast cell function, and excessive erythropoiesis. A targeted deletion of 65 amino acids in the N-terminal SH2 (SH2-N) domain of Shp-2 leads to an embryonic lethality at midgestation in homozygous mutant mice. To further dissect the Shp-2 function in hematopoietic development, we have isolated homozygous Shp-2 mutant embryonic stem (ES) cells. Significantly reduced hematopoietic activity was observed when the mutant ES cells were allowed to differentiate into embryoid bodies (EBs), compared to the wild-type and heterozygous ES cells. Further analysis of ES cell differentiation in vitro showed that mutation in the Shp-2 locus severely suppressed the development of primitive and definitive erythroid progenitors and completely blocked the production of progenitor cells for granulocytes-macrophages and mast cells. Reverse transcriptase PCR analysis of the mutant EBs revealed reduced expression of several specific marker genes that are induced during blood cell differentiation. Stem cell factor induction of mitogen-activated protein kinase activity was also blocked in Shp-2 mutant cells. Taken together, these results indicate that Shp-2 is an essential component and primarily plays a positive role in signaling pathways that mediate hematopoiesis in mammals. Furthermore, stimulation of its catalytic activity is not sufficient, while interaction via the SH2 domains with the targets or regulators is necessary for its biological functions in cells. The in vitro ES cell differentiation assay can be used as a biological tool in dissecting cytoplasmic signaling pathways.

Animals↗
PubMed1997

Complementary deoxyribonucleic acid cloning and characterization of a putative human axonemal dynein light chain gene.

Immotile Cilia Syndrome (ICS) is characterized by recurrent sinus and lung infections, bronchiectasis, and sperm immotility. Nasal cilia and sperm tails in patients with ICS exhibit a variety of ultrastructural defects, often including shortening or absence of the inner dynein arms. Immotile mutant strains of Chlamydomonas, a biflagellated algae, have ultrastructural defects similar to those seen in patients with this clinical disorder. Furthermore, splice-site mutations in the Chlamydomonas inner dynein arm gene (p28) are associated with impaired flagellar motility. We therefore hypothesized that the human homologue of the Clamydomonas dynein p28 gene would be an attractive candidate gene for patients with ICS. Accordingly, we cloned the full length complementary DNA (cDNA) and genomic clone by screening of appropriate libraries and databases, using the protein sequence of the Chlamydomonas p28 gene. The human homologue is encoded by a 921 bp transcript (accession no. AF006386) with an open reading frame of 257 amino acids. Using somatic cell and radiation hybrid panels, the hp28 gene was mapped to human chromosome 1p35.1. The hp28 cDNA probe hybridizes to sequences in all species on a zoo blot containing genomic DNA from yeast to human. Northern blot analysis reveals two hp28 gene transcripts, 0.9 and 2.5 kb, in many tissues. The 0.9 kb transcript is expressed at a 20-fold higher level than the 2.5-kb transcript in the testis. The entire gene is included in a 20-kb EcoRI genomic fragment and has 7 exons and 6 introns. Cloning of the hp28 cDNA and mapping of the intron-exon junctions should now make it possible to test whether a subset of ICS is a consequence of mutations in the human axonemal dynein light chain gene hp28.

Base Sequence↗
PubMed1997

[High-malar plasty through an intraoral approach].

Request for high-malar plasty, a purely aesthetic surgery, is fairly common in the Oriental. For an optimal result, consideration should be given to integrating the malar reconstruction with the facial features. A new method for correction of the high-malar deformity was introduced, in which the intraoral approach through an L-shaped incision and osteotomy were employed. The technique is simple, safe and can be done with local anesthesia on an outpatient basis. From December 1994 to February 1996, we have successfully treated 25 cases with this method. Postoperative follow-up for 6 months to 1 year revealed aesthetically satisfactory results. Complications are minor, including swelling, ecchymosis, etc. We conclude that high-malar plasty through the intraoral approach is a safe, effective and relatively simple technique for mild and moderate high-malar deformity.

Adult↗