Discovery of GCC5694A: A potent and selective sodium glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes.

Sodium-dependent glucose co-transporter 2 (SGLT2) has emerged as a promising drug target for the treatment of type 2 diabetes, and recently, several SGLT2 inhibitors have been approved for clinical use. A series of molecules with a C-aryl glucoside scaffold was designed and synthesized for biological evaluation. Among the molecules tested, a dihydrobenzofuran-containing analog, 14g (GCC5694A), exhibited excellentin vitro activity against SGLT2 (IC50 = 0.460 nM), good selectivity for SGLT1, and good metabolic stability. Data from further evaluation of the compound in animal models showed that this molecule is a promising candidate for development as an anti-diabetic agent.

High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production.

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 µmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 µmol/min/mg DCW and 1,992 µmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (P(H36)) was employed for the secretory production of endoxylanase in fed-batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum.

PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes.

Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+ and myofilament Ca2+ sensitivity. To examine the roles of PKC-δ and PKC-ε in these acute responses to ET-1, kinase inactive dominant negative PKC (dn-PKC) constructs were expressed in adult rat ventricular myocytes. Yellow fluorescent protein (YFP) was fused to dn-PKC constructs to visualize expression and localization of dn-PKC in living myocytes. Due to an alanine to glutamate mutation in the pseudosubstrate site, dn-PKCs constitutively translocated to anchoring sites and were unaffected by agonist or phorbol ester treatment. Dn-PKC-δ-YFP mainly distributed at Z-lines and at intercalated disks in adult myocytes, whereas dn-PKC-ε-YFP stained the surface sarcolemma, T-tubules/Z-lines and perinuclear region. Myocytes expressing dn-PKC-δ-YFP showed normal systolic Ca2+ and contractile responses to ET-1. In contrast, the entire ensemble of ET-1 responses was blocked in myocytes expressing dn-PKC-ε-YFP including increased Ca2+ transients, enhanced myofilament Ca2+ sensitivity, and positive inotropy. This report provides direct evidence that PKC-ε is activated early and robustly following ET-1 stimulation and thus mediates multiple intracellular changes underlying the acute actions of ET-1 on myocardium.

Endothelin receptor overexpression alters diastolic function in cultured rat ventricular myocytes.

The endothelin (ET) signaling pathway controls many physiological processes in myocardium and often becomes upregulated in heart diseases. The aim of the present study was to investigate the effects of ET receptor upregulation on the contractile function of adult ventricular myocytes. Primary cultured adult rat ventricular myocytes were used as a model system of ET receptor overexpression in the heart. Endothelin receptor type A (ETA) or type B (ETB) was overexpressed by Adenoviral infection, and the twitch responses of infected ventricular myocytes were measured after ET-1 stimulation. Overexpression of ETA exaggerated positive inotropic effect (PIE) and diastolic shortening of ET-1, and induced a new twitch response including twitch broadening. On the contrary, overexpression of ETB increased PIE of ET-1, but did not affect other two twitch responses. Control myocytes expressing endogenous receptors showed a parallel increase in twitch amplitude and systolic Ca(2+) in response to ET-1. However, intracellular Ca(2+) did not change in proportion to the changes in contractility in myocytes overexpressing ETA. Overexpression of ETA enhanced both systolic and diastolic contractility without parallel changes in Ca(2+). Differential regulation of this nature indicates that upregulation of ETA may contribute to diastolic myocardial dysfunction by selectively targeting myofi lament proteins that regulate resting cell length, twitch duration and responsiveness to prevailing Ca(2+).

Novel thiophenyl C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents.

Novel thiophene C-aryl glucoside SGLT2 inhibitors were designed and synthesized. Two different types of thiophene derivatives were readily prepared. Among the compounds tested, ethylphenyl at the distal ring 71p showed the best in vitro inhibitory activity in this series to date (IC(50)=4.47 nM) against SGLT2.

Findings from SHAZ!: a feasibility study of a microcredit and life-skills HIV prevention intervention to reduce risk among adolescent female orphans in Zimbabwe.

This study tested the feasibility of a combined microcredit and life-skills HIV prevention intervention among 50 adolescent female orphans in urban/peri-urban Zimbabwe. Quantitative and qualitative data were collected on intervention delivery, HIV knowledge and behavior, and economic indicators. The study also tested for HIV, HSV-2, and pregnancy. At 6 months, results indicated improvements in knowledge and relationship power. Because of the economic context and lack of adequate support, however, loan repayment and business success was poor. The results suggest that microcredit is not the best livelihood option to reduce risk among adolescent girls in this context.

Assessment of the Association of Chlamydia trachomatis Infection and Adverse Perinatal Outcomes with the Use of Population-Based Chlamydia Case Report Registries and Birth Records.

OBJECTIVE: We assessed the relationship between Chlamydia trachomatis (CT) infections identified during pregnancy and adverse perinatal birth outcomes (including premature rupture of membranes, preterm delivery, and low birthweight) by matching CT reports and birth records. METHODS: We merged California birth records from 1997, 1998, and 1999 with California CT reports from the same years to determine the proportion of birth records matched to a female CT report, using maternal last name, first name, date of birth, and county of residence. We used logistic regression to assess the crude and adjusted association between a CT report less than 10 months before the birth record date and premature rupture of membranes, preterm delivery, and low birthweight. These results were adjusted for age, race/ethnicity, level of education, and prenatal care. RESULTS: Of 675,786 birth records and 101,296 female CT reports, 14,039 women had a CT case report and a birth record; 10,917 birth records (1.6%) were matched to a CT report during pregnancy, and 10,940 (10.8%) of CT reports were matched to a birth record date 10 months after date of diagnosis/report. For premature rupture of membranes, the adjusted odds ratio (AOR) was 1.2, 95% confidence interval (CI) 1.0, 1.3; for low birthweight, the AOR was 1.2, 95% CI 1.1, 1.3. The reduction in birthweight associated with prenatal CT infection was 31.7 grams. CONCLUSIONS: The increased risk of adverse perinatal outcomes associated with prenatal CT infection supports current prenatal CT screening guidelines. Matching of surveillance and vital statistics data sources was an efficient method to assess this association.

Suppressive effects of bee venom on the immune responses in collagen-induced arthritis in rats.

The effect of bee venom (BVA) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. Male rats were immunized with an emulsion of 200 microg of CII and complete Freund's adjuvant (CFA). The rats were then given intraperitoneally (i.p.) injection of a suspension of BVA or saline during the experiment. The effect of BVA on cellular responses to CII was examined. In the control rats, the onset of arthritis was observed at the 24th day after the CII-immunization, and the severity of CIA was developed gradually. As compared with rats treated with saline, BVA i.p. injected at doses of more than 20 microl/100g mouse once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-1beta, -2, -6, tumor necrosis factor-alpha and interferon-gamma when the cells were obtained from rats 24 days after immunization and cultured in vitro with CII. When rats were injected i.p. with sheep red blood cells, hemagglutination titers in BVA-treated and control rats did not differ significantly when low doses of BVA was given to rats. However, i.p. injection of BVA at doses of more than 10 microl/100g/day suppressed antibody production. Pretreatment of rats with BVA could inhibit the development of collagen arthritis even when 10-20 microl/100g/day of the BVA were used for pretreatment. Interestingly, higher doses than 10 microlBVA/100g mouse were much effective for arthritis incidence. Treatment of rats with BVA prevented the development of collagen arthritis in a dose-dependent manner. Doses of BVA (15 and 20 microl/100g) resulted in decreased incidence of arthritis. In conclusion, therapeutic i.p injection with BVA improved the clinical course of the disease and the immune response to CII.

Female condom uptake and acceptability in Zimbabwe.

As the first phase of a two-phase prospective cohort study to assess the acceptability of the diaphragm as a potential HIV/STI prevention method, we conducted a 2-month prospective study and examined the effect of a male and female condom intervention on female condom (FC) use among 379 sexually active women in Harare, Zimbabwe. Reported use of FC increased from 1.1% at baseline to 70.6% at 2-month follow-up. Predictors of FC uptake immediately following the intervention included interest in using FC, liking FC better than male condoms, and believing one could use them more consistently than male condoms. Women reported 28.8% of sex acts protected by FC in the 2 weeks prior to last study visit. Though FC may not be the preferred method for the majority of women, with access, proper education, and promotion they may be a valuable option for some Zimbabwean women.

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes.

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.

G-protein coupled receptor signaling in myocardium: not for the faint of heart.

Catecholamines, endothelin-1 and angiotensin II are among a diverse group of diffusible extracellular signals that regulate pump function of the heart by binding to G-protein coupled receptors (GPCR). When the body demands a temporary boost of power output or if temporary budgeting of resources is required, these signals can adjust heart rate and contractile strength to maintain continuous perfusion of all vascular beds with nutrient- and oxygen-rich blood. Given adequate time in the face of prolonged challenges, activation of GPCRs can also promote "remodeling of the heart" by increasing cell size, organ size, and chamber dimensions, or by varying tissue composition and altering the expression of protein isoforms controlling excitability and contractility. A common feature of heart disease is the state of chronic activation of GPCR signaling systems. Paradoxically, whereas acute activation is beneficial, chronic activation often contributes to further deterioration of cardiac performance. A better understanding of how chronic GPCR activation contributes to the development of heart disease is needed so that it can be translated into better prevention and therapeutic strategies in the clinic.

The importance of discreet use of the diaphragm to Zimbabwean women and their partners.

We conducted a 6-month acceptability study of diaphragms as a potential HIV/STI prevention method among Zimbabwean women. We examined partner involvement in diaphragm use, and importance of discreet use (use without partner awareness). Of the 181 women who completed the study, 45% said discreet use was "very or extremely important" and in multivariate logistic regression, women were more likely to value discretion if their partners: had other partners; drank alcohol; or were believed to prefer condoms to diaphragms. Qualitative data confirmed these findings. Both women and their partners reported that diaphragms can be used discreetly and saw this as advantageous, for both sexual pleasure and female control. However, many were concerned that use without partner approval could lead to marital problems. Discreet use should be considered in development of barrier methods and in diaphragm promotion, if proven effective against HIV/STI.

Contraceptive intentions among adolescents after abortion.

Changes between choice of contraceptive methods before abortion and contraceptive intentions after abortion were assessed among 482 adolescents with regards to efficacy to prevent pregnancy or sexually transmitted diseases (STD) and human immunodeficiency virus (HIV). Adolescents substantially increased their intention to use oral contraceptives (214 of 452 who did not use before intended to after; p < .001) and depot-medroxyprogesterone acetate (DMPA) (121 of 469 who did not use before chose to after; p < .001). None of the 134 adolescents who used condoms as their primary contraceptive method before abortion intended to continue afterwards (p < .001). There was no difference in intention to use condoms after abortion among adolescents who received voluntary HIV counseling and testing compared to those who did not. Twenty-two percent of adolescents intended to use condoms together with spermicidal foam as their primary contraceptive method after abortion, thereby combining contraceptive efficacy with STD prevention. The intention to preferentially adopt hormonal methods as the primary contraceptive, especially among adolescents counseled and tested for HIV, is discouraging for STD and HIV prevention efforts in this adolescent population.

PICOT inhibits cardiac hypertrophy and enhances ventricular function and cardiomyocyte contractility.

Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca(2+) handing analysis revealed that increases in myofilament Ca(2+) responsiveness, together with increased rate of sarcoplasmic reticulum Ca(2+) reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.

Endothelin-1 and PKC induce positive inotropy without affecting pHi in ventricular myocytes.

It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.

Barrier method preferences and perceptions among Zimbabwean women and their partners.

In Zimbabwe, adult HIV prevalence is over 25% and acceptable prevention methods are urgently needed. Sixty-eight Zimbabwean women who had completed a barrier-methods study and 34 of their male partners participated in focus group discussions and in-depth interviews to qualitatively explore acceptability of male condoms, female condoms and diaphragms. Most men and about half of women preferred diaphragms because they are female-controlled and do not detract from sexual pleasure or carry stigma. Unknown efficacy and reuse were concerns and some women reported feeling unclean when leaving the diaphragm in for six hours following sex. Nearly half of women and some men preferred male condoms because they are effective and limit women's exposure to semen, although they reportedly detract from sexual pleasure and carry social stigma. Female condoms were least preferred because of obviousness and partial coverage of outer-genitalia that interfered with sexual pleasure.

Introducing diaphragms into the mix: what happens to male condom use patterns?

The objective of this analysis was to assess the effect of introducing the diaphragm on condom use patterns. Participants included one hundred eighty-nine women attending family planning clinics in Harare, Zimbabwe who reported less than 100% condom use. The proportion of acts where at least one method was used significantly increased over using follow-up; male condom use remained stable. A diaphragm was used with 50% to 54% of acts; male condoms were also used about 50% of the time. The proportion of acts where a female condom was used decreased. Women who used both male and female condoms were more likely to use diaphragms than those who reported not using female condoms. Introducing the diaphragm increased the overall proportion of protected acts. The proportion of acts where a male condom was used did not change. Female condoms use declined because concurrent use with the diaphragm is not possible.

Misoprostol compared with laminaria before early second-trimester surgical abortion: a randomized trial.

OBJECTIVE: To compare the efficacy and acceptability of same-day misoprostol and overnight laminaria for cervical ripening before early second-trimester surgical abortion. METHODS: We performed a randomized, double-blinded, controlled trial comparing 400 microg of vaginal misoprostol, given 3-4 hours preoperatively, with overnight laminaria before early second-trimester surgical abortion among women at 13.0-16.0 weeks of gestation (n = 84). The primary outcome was procedure time, and the sample size was based on 95% power to detect a difference of 4.5 minutes between groups. Secondary outcomes included completion of the procedure on the first attempt, procedural difficulty, and patients' pain scores and preferences. RESULTS: The average gestational duration was 14 weeks 6 days. Procedures performed after laminaria were significantly faster than those after misoprostol (median 3.4 versus 7.2 minutes, respectively, P = .01). Laminaria patients had significantly greater dilation than misoprostol patients at abortion (mean 43 versus 33 French, P < .001), and more misoprostol patients required additional dilation (85% versus 21%, P < .001). Physicians rated 27% of the misoprostol procedures as moderate-markedly difficult versus 5% of laminaria procedures (P = .01). Differences in efficacy were pronounced among nulliparous patients. There were no significant differences in ability to complete the procedure on the first attempt or patients' intraoperative pain scores. More women in the misoprostol group would choose their assigned method again (93% versus 62%, P < .01), and 82% of all subjects preferred a 1-day procedure. CONCLUSION: Early second-trimester abortions take longer and are technically more challenging after cervical ripening with same-day misoprostol than with overnight laminaria, but patients prefer same-day misoprostol.

HIV sentinel surveillance among women seeking elective pregnancy termination, San Francisco.

OBJECTIVE: The objective of this study was to measure HIV prevalence, HIV incidence, and risk factors for infection among women seeking elective pregnancy termination in San Francisco. STUDY: The authors conducted a cross-sectional survey comprising a consecutive sample of women seeking elective pregnancy termination in San Francisco's county hospital from August 2002 to July 2003. Demographic and risk behavior information was abstracted from routine clinic records. HIV testing was conducted on blood specimens collected for other purposes after removing identifying information. RESULTS: Based on 11 HIV-positives among 1,992 tested, HIV prevalence among women seeking pregnancy termination was 0.55% (95% confidence interval [CI], 0.28-0.99). One recent HIV seroconversion was detected for an annual incidence of 0.11% per year (95% CI, 0.23-0.88). In addition, risk factors significantly associated with HIV infection included sex with a known HIV-positive man, history of an abnormal Pap smear, history of genital herpes infection, history of trichomoniasis, and age 25 to 29 years. CONCLUSIONS: Women electing pregnancy termination can serve as a sentinel population to track trends in the HIV epidemic. However, barriers remain to wider implementation of the approach as a surveillance tool.