Stacy Weitsman - Academia.edu (original) (raw)
Papers by Stacy Weitsman
Digestive diseases and sciences, Jan 27, 2017
Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in... more Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in and differentiate irritable bowel syndrome with diarrhea (IBS-D) from other causes of diarrhea and healthy controls. To determine whether these antibodies can also diagnose and differentiate other IBS subtypes. Subjects with IBS-D based on Rome III criteria (n = 2375) were recruited from a large-scale multicenter clinical trial (TARGET 3). Healthy subjects without gastrointestinal (GI) diseases or symptoms (n = 43) and subjects with mixed IBS (IBS-M) (n = 25) or IBS with constipation (IBS-C) (n = 30) were recruited from two major medical centers. Plasma levels of anti-CdtB and anti-vinculin antibodies in all subjects were determined by enzyme-linked immunosorbent assay. Optical densities of ≥1.68 and ≥2.80 were considered positive for anti-vinculin and anti-CdtB, respectively. Plasma levels of anti-CdtB and anti-vinculin antibodies were highest in IBS-D and lowest in IBS-C and healthy c...
Gastroenterology and Hepatology, Jul 1, 2011
Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with an estimated... more Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with an estimated worldwide prevalence of 10-20%. IBS can be associated with severe gastrointestinal symptoms, including abdominal pain, bloating, and altered bowel function. Although the causes of IBS remain undefined, recent research has increasingly suggested roles for gut flora in IBS. These roles involve postinfectious IBS, which can occur after a single episode of acute gastroenteritis, and small intestinal bacterial overgrowth, in which elevated populations of aerobic and anaerobic bacteria cause abdominal pain and altered bowel function. More recently, potential roles for methanogens in contributing to IBS subtypes have also been identified. In this paper, we review the different mechanisms by which gut flora may contribute to IBS and also discuss the efficacy and safety of various antibiotic therapies for treating IBS symptoms.
Endocrinology, Jul 1, 2013
Biology of Reproduction, 1993
Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I... more Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating differentiation of the ovarian theca-interstitial cells (TIC) during early follicular development. IGF-I has been shown to synergistically enhance the stimulation of androgen biosynthesis and to increase LH binding in TIC. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on 3-hydroxysteroid dehydrogenase (30-HSD) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I for up to 6 days. At various times cytoplasmic RNA was extracted from the TIC, and 3-HSD mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Amplification of 3,-HSD mRNA using primers designed to distinguish between the type I and type II 3-HSD gene products revealed that the TIC expressed primarily the type I gene. Increasing concentrations of LH (0-1 g/ml) stimulated a dose-related increase in 3-HSD mRNA that was approximately 3fold at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) increased 3-HSD mRNA approximately 2-fold over TIC treated with LH alone. IGF-I alone also stimulated a dose-related increase in 3-HSD mRNA that reached a level approximately 3-fold greater than unstimulated levels. Addition of LH (100 ng/ml) increased 3-HSD mRNA at each concentration of IGF-I. Time-course studies revealed that expression of 3[-HSD mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone, or LH plus IGF-I and then declined at 4 and 6 days. More detailed studies demonstrated that LH stimulated a rapid increase in progesterone production by the TIC after an approximately 2-h lag. Progesterone levels began to decline after approximately 20 h in culture. Although IGF-I alone did not alter progesterone production, concomitant treatment with LH and IGF-I increased progesterone to levels approximately 2-fold higher than in TIC treated with LH alone without altering the temporal pattern of progesterone production. When expression of 3,-HSD mRNA was examined, LH stimulated an initial increase between 2 and 4 h and a secondary increase at approximately 30 h. During the first 30 h of culture, concomitant treatment with LH and IGF-I caused an approximately 3-fold increase in 3-HSD mRNA levels above the levels in TIC treated with LH alone, but did not change the LH-stimulated levels after 30 h. Interestingly, the time course of 3-HSD expression was much slower in response to IGF-I alone, rising after an approximately 20-h lag to maximum levels at 30 h. The results of our studies demonstrate that IGF-I stimulates the expression of 3,-HSD mRNA in ovarian TIC, supporting the hypothesis that IGF-I may play a role in stimulating TIC differentiation in developing follicles.
Endocrinology, Mar 1, 1997
During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian... more During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-dayold rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 ϫ 10 4 cells/well) were cultured with LH (0 -3 ng/ml) and/or HGF (0 -100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC 50 ϭ 1.5 Ϯ 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P 4 ) synthesis by TIC (1201 Ϯ 190 pg/ml) compared to that by control cells (210 Ϯ 30 pg/ml). HGF stimulated basal P 4 production, and LH-dependent P 4 synthesis was augmented 2.6-fold by HGF (ED 50 ϭ 0.3 Ϯ 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P450 17␣ hydroxylase/ C 17,20 lyase mRNA content was reduced 4.5-fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P 4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P450 17␣ hydroxylase/C 17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis. (Endocrinology 138: [691][692][693][694][695][696][697] 1997)
The Journal of Clinical Endocrinology and Metabolism, 2002
A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expres... more A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expression of estrogen receptors (ERs) could contribute to poor follicular development and ovulatory failure in polycystic ovary syndrome (PCOS). The aim of this study was to determine whether there are differences in ER␣ and ER expression in granulosa cells (GC) and theca cells (TC) from women with PCOS, compared with regularly cycling women. GC and TC were obtained by microdissection from 12 polycystic and 23 normal ovaries. ER␣ and ER mRNA and protein expression were measured by semiquantitative RT-PCR and Western blot, respectively. In control ovaries, both GC and TC ER␣ mRNAs were higher in small antral (SA) than in dominant follicles. ER␣ mRNA was similar in PCOS and size-matched control follicles. In control follicles, ER␣ protein concentrations were higher in GC than in TC. In GC, the ER␣ concentrations were comparable among SA, dominant, and PCOS follicles. In TC, ER␣ concentrations were lower in dominant follicles but were markedly increased in PCOS. In control ovaries, GC and TC expression of ER mRNA was higher in SA, compared with dominant follicles. In PCOS, ER mRNA was intermediate between SA and dominant follicles in both GC and TC. In GC, the ER protein concentrations followed the same pattern as mRNA expression; but in TC ER, protein in PCOS was equivalent to that in dominant follicles. The results of this study demonstrate that there are significant alterations in the expression of ER␣ and ER in PCOS that may be related to abnormal follicular development. (J Clin Endocrinol Metab 87: 5532-5538, 2002)
Molecular and Cellular Endocrinology, Oct 31, 1993
Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) ma... more Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theta-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P4.50 (P45Oscc > mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P45Oscc mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (O-1 pg/ml) stimulated a dose-related increase in P45Oscc mRNA (EDS,, = 36.2 f 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/mlI caused a small increase in P450,cc mRNA over TIC treated with LH alone but did not alter the ED,,, for LH stimulation. IGF-I alone also stimulated an increase in P450,cc mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P45Oscc mRNA (ED,,, = 1.2 f 0.05 ng/mll. Time-course studies revealed that expression of P45Oscc mRNA was greatest at 2 days in TIC treated with IGF-
Obesity, 2016
Methanogens colonizing the human gut produce methane and influence host metabolism. This study ex... more Methanogens colonizing the human gut produce methane and influence host metabolism. This study examined metabolic parameters in methane-producing subjects before and after antibiotic treatment. Eleven prediabetic methane-positive subjects (9F, 2M) with obesity (BMI 35.17 ± 7.71 kg/m(2) ) aged 47 ± 9 years were recruited. Subjects underwent breath testing, symptom questionnaire, oral glucose tolerance test (OGTT), lipid profile, and stool Methanobrevibacter smithii levels, gastric transit, and energy utilization analyses. After a 10-day antibiotic therapy (neomycin 500 mg bid/rifaximin 550 mg tid), all testing was repeated. Baseline stool M. smithii levels correlated with breath methane (R = 0.7, P = 0.05). Eight subjects (73%) eradicated breath methane and showed reduced stool M. smithii (P = 0.16). After therapy, methane-eradicated subjects showed significant improvements in low-density lipoprotein (LDL) (P = 0.028), total cholesterol (P = 0.01), and insulin levels on OGTT (P = 0.05 at 120 minutes), lower blood glucose levels on OGTT (P = 0.054 at 90 minutes), significant reductions in bloating (P = 0.018) and straining (P = 0.059), and a trend toward lower stool dry weight. No changes were detected in gastric emptying time or energy harvest. Breath methane eradication and M. smithii reduction are associated with significant improvements in total cholesterol, LDL, and insulin levels and with lower glucose levels in prediabetic subjects with obesity. The underlying mechanisms require further elucidation.
Journal of Virology
ABSTRACT
Journal of Virology
Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have r... more Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modffied the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-lJR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-lJRCsF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.
Journal of Virology
Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through... more Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.
Digestive diseases and sciences, Jan 27, 2017
Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in... more Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in and differentiate irritable bowel syndrome with diarrhea (IBS-D) from other causes of diarrhea and healthy controls. To determine whether these antibodies can also diagnose and differentiate other IBS subtypes. Subjects with IBS-D based on Rome III criteria (n = 2375) were recruited from a large-scale multicenter clinical trial (TARGET 3). Healthy subjects without gastrointestinal (GI) diseases or symptoms (n = 43) and subjects with mixed IBS (IBS-M) (n = 25) or IBS with constipation (IBS-C) (n = 30) were recruited from two major medical centers. Plasma levels of anti-CdtB and anti-vinculin antibodies in all subjects were determined by enzyme-linked immunosorbent assay. Optical densities of ≥1.68 and ≥2.80 were considered positive for anti-vinculin and anti-CdtB, respectively. Plasma levels of anti-CdtB and anti-vinculin antibodies were highest in IBS-D and lowest in IBS-C and healthy c...
Gastroenterology and Hepatology, Jul 1, 2011
Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with an estimated... more Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with an estimated worldwide prevalence of 10-20%. IBS can be associated with severe gastrointestinal symptoms, including abdominal pain, bloating, and altered bowel function. Although the causes of IBS remain undefined, recent research has increasingly suggested roles for gut flora in IBS. These roles involve postinfectious IBS, which can occur after a single episode of acute gastroenteritis, and small intestinal bacterial overgrowth, in which elevated populations of aerobic and anaerobic bacteria cause abdominal pain and altered bowel function. More recently, potential roles for methanogens in contributing to IBS subtypes have also been identified. In this paper, we review the different mechanisms by which gut flora may contribute to IBS and also discuss the efficacy and safety of various antibiotic therapies for treating IBS symptoms.
Endocrinology, Jul 1, 2013
Biology of Reproduction, 1993
Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I... more Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating differentiation of the ovarian theca-interstitial cells (TIC) during early follicular development. IGF-I has been shown to synergistically enhance the stimulation of androgen biosynthesis and to increase LH binding in TIC. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on 3-hydroxysteroid dehydrogenase (30-HSD) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I for up to 6 days. At various times cytoplasmic RNA was extracted from the TIC, and 3-HSD mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Amplification of 3,-HSD mRNA using primers designed to distinguish between the type I and type II 3-HSD gene products revealed that the TIC expressed primarily the type I gene. Increasing concentrations of LH (0-1 g/ml) stimulated a dose-related increase in 3-HSD mRNA that was approximately 3fold at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) increased 3-HSD mRNA approximately 2-fold over TIC treated with LH alone. IGF-I alone also stimulated a dose-related increase in 3-HSD mRNA that reached a level approximately 3-fold greater than unstimulated levels. Addition of LH (100 ng/ml) increased 3-HSD mRNA at each concentration of IGF-I. Time-course studies revealed that expression of 3[-HSD mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone, or LH plus IGF-I and then declined at 4 and 6 days. More detailed studies demonstrated that LH stimulated a rapid increase in progesterone production by the TIC after an approximately 2-h lag. Progesterone levels began to decline after approximately 20 h in culture. Although IGF-I alone did not alter progesterone production, concomitant treatment with LH and IGF-I increased progesterone to levels approximately 2-fold higher than in TIC treated with LH alone without altering the temporal pattern of progesterone production. When expression of 3,-HSD mRNA was examined, LH stimulated an initial increase between 2 and 4 h and a secondary increase at approximately 30 h. During the first 30 h of culture, concomitant treatment with LH and IGF-I caused an approximately 3-fold increase in 3-HSD mRNA levels above the levels in TIC treated with LH alone, but did not change the LH-stimulated levels after 30 h. Interestingly, the time course of 3-HSD expression was much slower in response to IGF-I alone, rising after an approximately 20-h lag to maximum levels at 30 h. The results of our studies demonstrate that IGF-I stimulates the expression of 3,-HSD mRNA in ovarian TIC, supporting the hypothesis that IGF-I may play a role in stimulating TIC differentiation in developing follicles.
Endocrinology, Mar 1, 1997
During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian... more During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-dayold rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 ϫ 10 4 cells/well) were cultured with LH (0 -3 ng/ml) and/or HGF (0 -100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC 50 ϭ 1.5 Ϯ 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P 4 ) synthesis by TIC (1201 Ϯ 190 pg/ml) compared to that by control cells (210 Ϯ 30 pg/ml). HGF stimulated basal P 4 production, and LH-dependent P 4 synthesis was augmented 2.6-fold by HGF (ED 50 ϭ 0.3 Ϯ 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P450 17␣ hydroxylase/ C 17,20 lyase mRNA content was reduced 4.5-fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P 4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P450 17␣ hydroxylase/C 17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis. (Endocrinology 138: [691][692][693][694][695][696][697] 1997)
The Journal of Clinical Endocrinology and Metabolism, 2002
A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expres... more A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expression of estrogen receptors (ERs) could contribute to poor follicular development and ovulatory failure in polycystic ovary syndrome (PCOS). The aim of this study was to determine whether there are differences in ER␣ and ER expression in granulosa cells (GC) and theca cells (TC) from women with PCOS, compared with regularly cycling women. GC and TC were obtained by microdissection from 12 polycystic and 23 normal ovaries. ER␣ and ER mRNA and protein expression were measured by semiquantitative RT-PCR and Western blot, respectively. In control ovaries, both GC and TC ER␣ mRNAs were higher in small antral (SA) than in dominant follicles. ER␣ mRNA was similar in PCOS and size-matched control follicles. In control follicles, ER␣ protein concentrations were higher in GC than in TC. In GC, the ER␣ concentrations were comparable among SA, dominant, and PCOS follicles. In TC, ER␣ concentrations were lower in dominant follicles but were markedly increased in PCOS. In control ovaries, GC and TC expression of ER mRNA was higher in SA, compared with dominant follicles. In PCOS, ER mRNA was intermediate between SA and dominant follicles in both GC and TC. In GC, the ER protein concentrations followed the same pattern as mRNA expression; but in TC ER, protein in PCOS was equivalent to that in dominant follicles. The results of this study demonstrate that there are significant alterations in the expression of ER␣ and ER in PCOS that may be related to abnormal follicular development. (J Clin Endocrinol Metab 87: 5532-5538, 2002)
Molecular and Cellular Endocrinology, Oct 31, 1993
Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) ma... more Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theta-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P4.50 (P45Oscc > mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P45Oscc mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (O-1 pg/ml) stimulated a dose-related increase in P45Oscc mRNA (EDS,, = 36.2 f 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/mlI caused a small increase in P450,cc mRNA over TIC treated with LH alone but did not alter the ED,,, for LH stimulation. IGF-I alone also stimulated an increase in P450,cc mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P45Oscc mRNA (ED,,, = 1.2 f 0.05 ng/mll. Time-course studies revealed that expression of P45Oscc mRNA was greatest at 2 days in TIC treated with IGF-
Obesity, 2016
Methanogens colonizing the human gut produce methane and influence host metabolism. This study ex... more Methanogens colonizing the human gut produce methane and influence host metabolism. This study examined metabolic parameters in methane-producing subjects before and after antibiotic treatment. Eleven prediabetic methane-positive subjects (9F, 2M) with obesity (BMI 35.17 ± 7.71 kg/m(2) ) aged 47 ± 9 years were recruited. Subjects underwent breath testing, symptom questionnaire, oral glucose tolerance test (OGTT), lipid profile, and stool Methanobrevibacter smithii levels, gastric transit, and energy utilization analyses. After a 10-day antibiotic therapy (neomycin 500 mg bid/rifaximin 550 mg tid), all testing was repeated. Baseline stool M. smithii levels correlated with breath methane (R = 0.7, P = 0.05). Eight subjects (73%) eradicated breath methane and showed reduced stool M. smithii (P = 0.16). After therapy, methane-eradicated subjects showed significant improvements in low-density lipoprotein (LDL) (P = 0.028), total cholesterol (P = 0.01), and insulin levels on OGTT (P = 0.05 at 120 minutes), lower blood glucose levels on OGTT (P = 0.054 at 90 minutes), significant reductions in bloating (P = 0.018) and straining (P = 0.059), and a trend toward lower stool dry weight. No changes were detected in gastric emptying time or energy harvest. Breath methane eradication and M. smithii reduction are associated with significant improvements in total cholesterol, LDL, and insulin levels and with lower glucose levels in prediabetic subjects with obesity. The underlying mechanisms require further elucidation.
Journal of Virology
ABSTRACT
Journal of Virology
Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have r... more Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modffied the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-lJR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-lJRCsF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.
Journal of Virology
Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through... more Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.