Biomedical subjects

C Ko

Publications and source records attributed to C Ko.

At least 19 recordsLinked to original sources

PubMed2005

Inhaled fluticasone in bronchiectasis: a 12 month study.

BACKGROUND: The clinical efficacy of inhaled corticosteroid (ICS) treatment has not been evaluated in bronchiectasis, despite the presence of chronic airway inflammation. METHODS: After three consecutive weekly visits, 86 patients were randomised to receive either fluticasone 500 mug twice daily (n = 43, 23F, mean (SD) age 57.7 (14.4) years) or matched placebo (n = 43, 34F, 59.2 (14.2) years) and reviewed regularly for 52 weeks in a double blind fashion. RESULTS: 35 and 38 patients in the fluticasone and placebo groups completed the study. Significantly more patients on ICS than on placebo showed improvement in 24 hour sputum volume (OR 2.5, 95% CI 1.1 to 6.0, p = 0.03) but not in exacerbation frequency, forced expiratory volume in 1 second, forced vital capacity, or sputum purulence score. Significantly more patients with Pseudomonas aeruginosa infection receiving fluticasone showed improvement in 24 hour sputum volume (OR 13.5, 95% CI 1.8 to 100.2, p = 0.03) and exacerbation frequency (OR 13.3, 95% CI 1.8 to 100.2, p = 0.01) than those given placebo. Logistic regression models revealed a significantly better response in sputum volume with fluticasone treatment than with placebo among subgroups of patients with 24 hour sputum volume <30 ml (p = 0.04), exacerbation frequency </=2/year (p = 0.04), and sputum purulence score >5 (p = 0.03). CONCLUSIONS: ICS treatment is beneficial to patients with bronchiectasis, particularly those with P. aerurginosa infection.

Administration, Inhalation↗

PubMed2005

A prediction rule for clinical diagnosis of severe acute respiratory syndrome.

A prospective study was undertaken to identify clinical, radiographical, haematological and biochemical profiles of severe acute respiratory syndrome (SARS) patients. A prediction rule, which demarcates low from high risk patients for SARS in an outbreak situation was developed. A total of 295 patients with unexplained respiratory illnesses, admitted to Queen Mary Hospital, Hong Kong SAR, China, in March to July 2003, were evaluated for clinical, radiological, haematological and alanine transaminase (ALT) data daily for 3 days after hospitalisation. In total, 44 cases were subsequently confirmed to have SARS by RT-PCR (68.2%) and serology (100%). The scoring system of attributing 11, 10, 3, 3 and 3 points to the presence of independent risk factors, namely: epidemiological link, radiographical deterioration, myalgia, lymphopenia and elevated ALT respectively, generated high and low-risk (total score 11-30 and 0-10, respectively) groups for SARS. The sensitivity and specificity of this prediction rule in positively identifying a SARS patient were 97.7 and 81.3%, respectively. The positive and negative predictive values were 47.8 and 99.5%, respectively. The prediction rule appears to be helpful in assessing suspected patients with severe acute respiratory syndrome at the bedside, and should be further validated in other severe acute respiratory syndrome cohorts.

Adult↗

PubMed2004

Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme and a single-chain Fv antibody that targets CCR5.

The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have reduced or absent CCR5 are apparently otherwise healthy, but are resistant to HIV infection. With the goal of reducing CCR5 and protecting CCR5+ cells from R5-tropic HIV, we used Tag-deleted SV40-derived vectors to deliver several anti-CCR5 transgenes: 2C7, a single-chain Fv (SFv) antibody; VCKA1, a hammerhead ribozyme; and two natural CCR5 ligands, MIP-1alpha and MIP-1beta, modified to direct these chemokines, and hence their receptor to the endoplasmic reticulum. These transgenes were delivered using recombinant, Tag-deleted SV40-derived vectors to human CCR5+ cell lines and primary cells: monocyte-derived macrophages and brain microglia. All transgenes except MIP-1alpha decreased CCR5, as assayed by immunostaining, Northern blotting, and cytofluorimetry (FACS). Individually, all transgenes except MIP-1alpha protected from low challenge doses of HIV. At higher dose HIV challenges, protection provided by all transgenes diminished, the SFv and the ribozyme being most potent. Vectors carrying these two transgenes were used sequentially to deliver combination anti-CCR5 genetic therapy. This approach gave approximately additive reduction in CCR5, as measured by FACS and protected from higher dose HIV challenges. Reducing cell membrane CCR5 using anti-CCR5 transgenes, alone or in combinations, may therefore provide a degree of protection from R5-tropic strains of HIV.

Cell Line↗

PubMed2004

Pentaglobin in steroid-resistant severe acute respiratory syndrome.

SETTING: The treatment of severe acute respiratory syndrome (SARS) is at best controversial, although there is considerable anecdotal experience to show the benefits of corticosteroid therapy for selected patients. Some patients deteriorate relentlessly despite treatment with antibiotic, corticosteroid and mechanical ventilation. OBJECTIVE: To attempt to determine the clinical efficacy of pentaglobin, an IgM-enriched immunoglobulin preparation, on 12 severe SARS patients who continued to deteriorate despite corticosteroid and ribavirin therapy. DESIGN: Retrospective analysis of daily quantitative and radiographic data on the cohort in a regional teaching hospital. RESULTS AND CONCLUSION: There was significant improvement in radiographic scores, when compared with day 1, on days 5, 6 and 7 (P < 0.05) after commencement of pentaglobin treatment. Similarly, there was significant improvement in oxygen requirement, when compared with day 1, on days 6 and 7 (P < 0.05) after commencement of pentaglobin treatment. There were no reported adverse events attributable to pentaglobin administration. Ten patients made an uneventful recovery after treatment. One elderly man died from cardiorespiratory arrest despite clinical and radiological improvement, and another patient is making good progress. Pentaglobin is safe and probably effective in the treatment of steroid-resistant SARS. A double-blind placebo-controlled study should therefore be considered.

Adrenal Cortex Hormones↗

PubMed2004

Exhaled nitric oxide in bronchiectasis: the effects of inhaled corticosteroid therapy.

SETTING: While exhaled nitric oxide (eNO) levels are reduced by inhaled corticosteroid therapy in asthma, such treatment effect is unclear in bronchiectasis. DESIGN: Stable non-smoking bronchiectasis patients were randomised to receive either fluticasone (1 mg/daily) or identical placebo via the Accuhaler device. RESULTS: Sixty non-smoking patients (38 women; mean age 56.4 +/- 12.7 years) were recruited. Of these, half received inhaled fluticasone and half placebo therapy. eNO was measured using a chemiluminescence analyser at 0, 4, 12, 24, 36 and 52 weeks. There was no significant difference in eNO levels between fluticasone and placebo patients over the study period. There was no correlation between baseline eNO with age, FEV1, FVC, 24 h sputum volume or number of bronchiectatic segments. Patients with Pseudomonas aeruginosa (PA) infection, but not their counterparts, displayed a correlation between 0- and 52-week eNO levels. PA infection was associated with significantly lower eNO levels among the patients. CONCLUSIONS: Inhaled fluticasone therapy, despite being an effective anti-inflammatory agent, has no significant effect on eNO production, either at individual time points or over the entire 52-week profile, in bronchiectasis. It appears that eNO might not reflect the extent of airway inflammation in bronchiectasis.

Administration, Inhalation↗

PubMed2001

Post-transcriptional stimulation of the assembly and secretion of triglyceride-rich apolipoprotein B lipoproteins in a mouse with selective deficiency of brown adipose tissue, obesity, and insulin resistance.

A mouse model of insulin resistance and its associated dyslipidemia was generated by crossing mice expressing human apolipoprotein B (apoB) with mice lacking only brown adipose tissue (BATless). On a high fat diet, male apoB/BATless mice became obese, hypercholesterolemic, hypertriglyceridemic, and hyperinsulinemic compared with control apoB mice. Fast performance liquid chromatography revealed increased triglyceride concentrations in intermediate density lipoprotein/low density lipoprotein (LDL) and reduced high density lipoprotein cholesterol concentrations. Inhibition of lipolysis by the drug, tetrahydrolipostatin, demonstrated that very low density lipoprotein-sized particles were initially secreted. Metabolic studies employing Triton WR-1339 and either [(3)H]glycerol or [(3)H]palmitate showed that the hypertriglyceridemia in apoB/BATless mice was due to the increased synthesis and secretion of triglyceride. Furthermore, lipoprotein lipase and hepatic lipase activities were not defective. ApoB was also secreted at increased rates in the apoB/BATless mice. Similar levels of apoB mRNA in apoB and apoB/BATless mice indicated that apoB secretion was regulated post-transcriptionally. LDL receptor mRNA was increased in the apoB/BATless mice, indicating that the observed increase in apoB-lipoprotein secretion was not due to their decreased reuptake. Finally, mRNA levels of the large subunit of microsomal triglyceride transfer protein, a required component for very low density protein assembly, were not different between apoB and apoB/BATless mice. This rodent model should prove useful in exploring mechanisms underlying the regulation of apoB secretion in the context of insulin resistance.

Adipose Tissue, Brown↗

PubMed2001

Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues.

Investigation of the role of the second, more recently described estrogen receptor, denoted ERbeta, will be critical in understanding the molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERbeta in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ERbeta protein, due, in part, to the limited availability of human ERbeta-specific antibodies. Thus, our aim was to generate specific antibodies to human ERbeta and use them to determine the tissue-specific distribution and size(s) of the ERbeta protein. To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ERbeta. The antibodies, made in mice against human ERbeta amino acids 256-505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (F12) and were determined to be the IgG gamma1 isotype for E12, and IgG gamma2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ERbeta protein by Western blot analyses, and all failed to detect ERalpha. A9 and F12 were able to immunoprecipitate efficiently the native form of ERbeta protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ERbeta protein in granulosa cells of the rat ovary. Nuclear ERbeta is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62-64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ERbeta protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ERbeta in the human and in several other mammalian species.

Animals↗

PubMed2001

Coronary revascularization with or without cardiopulmonary bypass in patients with preoperative nondialysis-dependent renal insufficiency.

BACKGROUND: Preoperative renal insufficiency is a predictor of acute renal failure in patients undergoing conventional coronary artery bypass grafting. Off-pump coronary artery bypass operations have been shown to reduce renal dysfunction in patients with normal renal function, but the effect of this technique in patients with preoperative nondialysis-dependent renal insufficiency is unknown. METHODS: From June 1996 to December 1999, data of 3,250 consecutive patients undergoing coronary artery bypass grafting were prospectively entered into the Patient Analysis & Tracking Systems (PATS, Dendrite Clinical Systems, London, UK). Two hundred and fifty-three patients with preoperative serum creatinine more than 150 micromol/L were identified (202 patients on-pump, 51 patients off-pump), and clinical outcomes were analyzed. Serum creatinine and urea, in-hospital mortality, and morbidity were compared between groups. The association of perioperative factors with acute renal failure was investigated by multiple logistic regression analysis. RESULTS: Preoperative characteristics were similar between the groups. Mean number of grafts was 2.9 +/- 0.8 and 2.3 +/- 0.8 in the on-pump and off-pump groups, respectively (p < 0.0001). Comparison between groups showed a significantly higher incidence of stroke, inotropic requirement, blood loss, and transfusion of red packed cell and platelets in the on-pump group (all p < 0.05). Postoperative serum creatinine and urea were higher in the on-pump group with a significant difference at 12 hours postoperatively (p < 0.05). Logistic regression analysis identified cardiopulmonary bypass, serum creatinine level 60 hours postoperatively, inotropic requirement, need for intraaortic balloon pump, transfusion of red packed cell, and hours of ventilation as predictors of postoperative acute renal failure. CONCLUSIONS: This study suggests that off-pump coronary artery bypass operations reduce in-hospital morbidity and the likelihood of acute renal failure in patients with preoperative nondialysis-dependent renal insufficiency undergoing myocardial revascularization.

Acute Kidney Injury↗

PubMed2001

Impact of mentor guidance in surgical career selection.

PURPOSE: The aim of this study was to survey graduates of a university general surgical residency training program to determine factors that influenced their selection of a specialty field. METHODS: A 39-item questionnaire was mailed to 86 graduates of a university general surgery program who matriculated from 1975 to 1989. The impact of lifestyle, technology, clinical opportunity, and mentor guidance in the residents' selection of a specialty field and eventual clinical practice was assessed. Results were analyzed using Fisher's Exact test with significance determined at P less than.05. RESULTS: The response rate was 65% (56 of 86). Eighty percent of respondents identified the most important aspect influencing their choice of specialty was interest in that field; additional factors included perception of prestige, presence of clinical opportunity, mentor influence, and family priorities. Sixty-six percent of respondents chose the same career as their mentor, attributing this to the mentor's skill (n = 36, 68%), achievements (n = 35, 66%), and verbal recommendations about their specialty field (n = 24, 45%), p < 0.05. CONCLUSIONS: Surgical residents use many criteria in selecting a field of specialty with mentor guidance an important component. Knowledge of these influential areas should help training programs offer appropriate career guidance. J Pediatr Surg 36:1802-1804.

Adult↗

PubMed2001

Two novel quantitative trait loci on mouse chromosomes 6 and 4 independently and synergistically regulate plasma apoB levels.

An elevated plasma apolipoprotein B (apoB) level is a strong predictor of atherosclerosis and coronary heart disease. Epidemiologic and family linkage studies have suggested a genetic basis for the wide variations of plasma apoB levels in the general population. Using a human apoB transgenic (HuBTg) mouse model, we have previously shown that hepatic apoB-100 secretion is a major determinant of the high and low plasma human apoB levels in HuBTg mice of the C57BL/6 (B6) and 129/Sv (129) strains, respectively. In the present article, we present the identification of two novel quantitative trait loci (QTL) as major regulators of plasma human apoB levels in the F(2) and N(2) (backcrossed) offspring (n = 572) derived from crosses between the B6 and 129 mouse strains. These loci were designated ApoB regulator genes (Abrg), because the gene products are likely to be involved in the regulation of plasma apoB levels either directly or indirectly. The first locus, designated Abrg1, was mapped to chromosome 6 in 8-week-old male and female mice with a combined logarithm of odds ratio (LOD) score of 14 at the D6Mit55 marker ( approximately 45.9 cM). Abrg1 contributed approximately 35% of the genetic variance. The second locus, designated Abrg2, was mapped to chromosome 4 with an LOD score of 8.6 in 8-week-old male mice but an LOD score of only 2.0 in 8-week-old female mice at the D4Mit27 marker ( approximately 35 cM). Abrg2 contributed approximately 26% of the genetic variance. Epistasis between Abrg1 and Abrg2 was detected and accounted for approximately 12% of the genetic variance. The combination of these two QTL has major effects (>70%) on the regulation of plasma human apoB levels in the tested population. In summary, we have identified two novel loci that have a major role in the regulation of plasma apoB levels and are likely to regulate the secretory pathway of apoB. The human orthologs for the Abrg loci are strong candidates for human disorders characterized by altered plasma apoB levels, such as FCHL and familial hypobetalipoproteinemia.

Analysis of Variance↗

PubMed2000

Granulysin, a T cell product, kills bacteria by altering membrane permeability.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Amino Acid Sequence↗

PubMed2000

Expression of an intracisternal A-particle-like element in rat ovary.

We have isolated a rat intracisternal-A particle element (IAP)-like element (IAP-LE) from ovarian granulosa cells that appears to be identical to the rat EST clone AA964260. The compiled cDNA sequences contain several putative in-frame translation initiation codons with the largest capable of encoding a 365 amino acid protein with a reverse transcriptase domain in the N-terminus as well as a bipartite nuclear localization signal sequence in the middle. Northern blotting shows a major approximately 7 Kb transcript and a minor approximately 5 Kb transcript that are abundantly expressed in the ovary. In situ hybridization histochemistry using ovaries from gonadotropin-treated immature rats and regularly cycling adult rats show that this transcript is predominantly localized to granulosa cells of all healthy follicles, including primary follicles, and to newly-formed and healthy corpora lutea. This cell-specific expression pattern of the IAP-LE gene is distinct from those of the several known retroviral elements, suggesting the potentially novel functional importance of the IAP-LE gene. Taken together, our results demonstrate abundant and cell-specific expression of a novel IAP-LE in rat granulosa cells.

Amino Acid Sequence↗

PubMed2000

Progesterone receptor activation mediates LH-induced type-I pituitary adenylate cyclase activating polypeptide receptor (PAC(1)) gene expression in rat granulosa cells.

We have previously reported that the pituitary adenylate cyclase activating polypeptide (PACAP) gene is regulated in ovarian granulosa cells by the autocrine and/or paracrine interaction between progesterone and its nuclear receptor progesterone receptor (PR). To initiate studies on the functional significance of the progesterone-induced PACAP production in luteinizing granulosa cells, we sought to determine the expression and hormonal regulation of PACAP receptors in the rat ovary. The relative mRNA levels of three known PACAP receptor subtypes (PAC(1), VPAC(1), and VPAC(2)) were determined in ovaries of immature rats treated with gonadotropins, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Results show that all PAC(1), VPAC(1), and VPAC(2) transcripts are expressed at a detectable level in immature rat ovaries. Importantly, the ovarian level of PAC(1), but not VPAC(1) or VPAC(2), mRNA notably changes during gonadotropin challenges. Ovarian PAC(1) mRNA expression decreases during the pregnant mare's serum gonadotropin (PMSG)-induced follicular phase but substantially increases during the human chorionic gonadotropin (hCG)-induced periovulatory period. Because the hCG-induced increase in ovarian PAC(1) mRNA expression is attributable to the hormone-induced PAC(1) mRNA expression in granulosa cells of the preovulatory follicles, we next examined whether hCG regulates PAC(1) mRNA expression by directly acting on granulosa cells. When granulosa cells isolated from PMSG (40 h)-primed immature rats were challenged with hCG (or forskolin), PAC(1), but not VPAC(1) or VPAC(2), mRNA expression significantly increased within 6 h. Because the LH-induced PAC(1) mRNA expression (6 h) proceeds PR activation (3 h) in granulosa cells as the LH-induced PACAP mRNA expression (6 h) does, we further determined the cause-effect relationship among LH, PR activation and PAC(1) receptor gene expression, by examining the effect of PR antagonist, ZK98299, on the ability of LH to increase PAC(1) mRNA levels in luteinizing granulosa cells. Results show that ZK98299 inhibited the stimulatory effect of hCG (or forskolin) on PAC(1) mRNA expression, at the level of all known splice variants of PAC(1) mRNA in granulosa cells. In summary, our results demonstrating that PR activation is critical for the LH-induced PAC(1) gene expression in luteinizing granulosa cells suggest that PR activation regulates the finely tuned expression of the PACAP/PACAP receptor genes in luteinizing granulosa cells and thus dictates the timing of the autocrine and/or paracrine function of PACAP in preovulatory follicles.

Animals↗

PubMed2000

A comparison of two bedside tests for the detection of salicylates in urine.

OBJECTIVE: Ferric chloride (FC) and Trinder reagent (TR) have both been used to identify salicylates (ASA) in the urine of patients presenting with possible drug overdose. The authors sought to compare the sensitivities and specificities of these two reagents in detecting ASA in the urine of patients presenting to an emergency department (ED) with suspected drug overdose. METHODS: Patients were eligible for inclusion in this study if they presented to the ED with either suspected overdose or unexplained metabolic acidosis. One milliliter of the patient's urine was added to 1 mL of each of the two reagents. A positive test was defined as any darkening of the color of the reagent. Each patient had a quantitative serum ASA measured. RESULTS: Twenty of 180 patients (11%) had quantitative serum ASA levels above 5 mg/dL. Both reagents were 100% sensitive in identifying these patients. The specificity of FC was 71% compared with 73% for TR. The two reagents gave similar results in 91% of cases. CONCLUSION: Both FC and TR are reliable in detecting ASA in the urine of patients presenting with suspected drug overdose. A negative result with either test eliminates the need for a quantitative serum ASA level. Because FC has a longer shelf life than TR, it is the more practical reagent for use in the ED.

Adolescent↗

PubMed1999

Exposure to antibiotics induces expression of the Mycobacterium tuberculosis sigF gene: implications for chemotherapy against mycobacterial persistors.

The sigF gene encodes an alternate sigma factor found in Mycobacterium tuberculosis and related pathogenic mycobacteria. Determination of conditions of sigF expression is an important step in understanding the conditional gene regulation which may govern such processes as virulence and dormancy in mycobacteria. We constructed an in-frame translational lacZ-kan fusion within the sigF gene to determine the conditions of sigF expression. This reporter construct was expressed from a multicopy plasmid in a strain of BCG harboring an integrated luciferase reporter gene under the control of the mycobacteriophage L5 gp71 promoter. Antibiotic exposure, in particular, ethambutol, rifampin, streptomycin, and cycloserine treatment, increased the level of SigF reporter specific expression in a dose-dependent fashion. The level of SigF reporter specific expression increased over 100-fold in late-stationary-phase growth compared to that in exponential growth. During the exponential phase, SigF specific expression could be induced by a number of other stresses. Anaerobic metabolism induced SigF by greater than 150-fold, particularly in the presence of metronidazole. Cold shock increased the level of SigF specific expression, while heat shock decreased it. Oxidative stress was also an important inducer of SigF specific expression; a greater induction was seen with cumene hydroperoxide than with hydrogen peroxide. Comparisons of bacterial viability as determined by the luciferase assay or by plating serial dilutions revealed that luciferase gp71-dependent activity was an unreliable predictor of the numbers of CFU during stationary-phase growth and anaerobic metabolism. The induction of sigF following antibiotic exposure suggests that this bacterial transcription factor may control genes which are important for mycobacterial persistence in the host during chemotherapy.

Anaerobiosis↗

PubMed1999

The Staphylococcus aureus rsbW (orf159) gene encodes an anti-sigma factor of SigB.

SigB, a newly discovered alternative sigma factor of Staphylococcus aureus, has been shown to play an important role in stress responses and the regulation of virulence factors. The rsbW (orf159) gene is immediately upstream of sigB. Its gene product is homologous to Bacillus subtilis RsbW which under appropriate conditions binds to B. subtilis SigB and functions as an anti-sigma factor or negative posttranslational regulator. To define the function of S. aureus RsbW, both the S. aureus SigB and RsbW proteins were expressed in Escherichia coli and purified. Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB. In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the sar P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent. We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro. These results show that RsbW is a protein sequestering anti-sigma factor of S. aureus SigB and suggest that SigB activity in S. aureus is regulated posttranslationally.

Bacterial Proteins↗

PubMed1999

Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary.

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semi-quantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells.

Animals↗

PubMed1999

Genetic control of hepatic apoB-100 secretion in human apoB transgenic mouse strains.

Plasma apolipoprotein B (apoB) levels vary widely in the general population and elevated plasma levels of apoB are associated with higher risk for atherosclerotic coronary heart disease. Determination of genetic factors regulating population variance of plasma apoB levels is complicated by the genetic heterogeneity of human populations. Using a congenic human apoB transgenic mouse strain in the C57BL/6 background (B6 HuBTg), we assessed genetic effects on the variance of plasma apoB, and on hepatic apoB-100 secretion rates. Six inbred mouse strains were crossed with the B6 HuBTg strain. Mean plasma apoB levels in the parental B6 HuBTg strain were 95 +/- 14 mg/dl. F1 human apoB transgenic offspring displayed plasma human apoB levels ranging from 60 to 105 mg/dl. In three F1 strains, the BALB/B6, C3H/B6 and 129/B6 strains, markedly lower plasma apoB levels (61 +/- 11, 64 +/- 5, and 67 +/- 8 mg/dl) were due to lower apoB-100 secretion rates. Human apoB mRNA levels in these three F1 strains were similar to those of the parental B6 strain suggesting that the mechanism for varying apoB secretion rates is most likely not transcriptional. In summary, we have identified three inbred mouse strains possessing polymorphic alleles which, when crossed with the B6 strain, lower plasma apoB levels and apoB-100 secretion in their F1 offspring. These mouse strains provide a powerful tool for genetic analysis of factors regulating apoB-100 secretion and hence plasma apoB levels.

Alleles↗