Osteoporosis epidemiology review and panacea osteoporosis evaluation study.
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Adult↗
Biomedical subjects
S Rao
Publications and source records attributed to S Rao.
Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol.
Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.
Animals↗
Quantitation of cerivastatin and its seven acid and lactone biotransformation products in human serum by liquid chromatography-electrospray tandem mass spectrometry.
A method for the simultaneous quantitation of cerivastatin (acid) and its biotransformation products, cerivastatin lactone, M-1 (acid), M-1 lactone, M-23 (acid), M-23 lactone, M-24 (acid) and M-24 lactone, in human serum by high-performance liquid chromatography (LC) with positive ion electrospray tandem mass spectrometry (MS-MS) was developed and validated. The method involves extraction of cerivastatin and its biotransformation products from acidified human serum (0.5 ml) using methyl tert.-butyl ether. The standard curve ranges in human serum were from 0.0100 to 10.0 ng/ml for cerivastatin and cerivastatin lactone, 0.0500 to 10.0 ng/ml for M-1 (acid) and M-1 lactone, 0.100 to 10.0 ng/ml for M-23 (acid) and M-23 lactone, and 0.500 to 10.0 ng/ml for M-24 (acid) and M-24 lactone. The lactone compounds in human serum at room temperature underwent considerable conversion to the corresponding acid compounds after only 4 h. Lowering the serum pH with a pH 5.0 buffer stabilized the lactone compounds for up to 24 h at room temperature. The degree of lactonization of the acid compounds was < or = 3.5% and the degree of hydrolysis of the lactone compounds was < or = 6.0% during the entire assay procedure. All the eight analytes eluted within 2.0 min and the total run time was only 3.5 min.
Biotransformation↗
The Ets factors PU.1 and Spi-B regulate the transcription in vivo of P2Y10, a lymphoid restricted heptahelical receptor.
To investigate the in vivo functions of PU.1 and Spi-B, two highly related Ets transcription factors, we previously generated PU. 1(+/+)Spi-B(-/-) and PU.1(+/-)Spi-B(-/-) mice and demonstrated a significant decrease in B-cell receptor (BCR) signaling in mutants. Major components of BCR signaling appear to be expressed at normal levels in these mice, implying that PU.1 and Spi-B cooperate in the transcription of additional target genes important for antigen receptor signaling. We used subtractive hybridization to identify novel in vivo PU.1/Spi-B target genes and determined that the expression of a heptahelical receptor, P2Y10, is dramatically reduced in PU.1(+/-)Spi-B(-/-) B-cells. Further analysis shows that P2Y10 expression is restricted to lymphoid cells and parallels that of Spi-B in B-lymphocytes. Lastly, the P2Y10 promoter contains a PU. 1/Spi-B binding site functionally required for efficient transcription in B-cells. Thus, P2Y10 is likely to be a direct in vivo transcriptional target for PU.1 and Spi-B and provides a unique model to explore transcriptional regulation by this Ets factor subfamily. Furthermore, P2Y10 suggests an intriguing connection between heterotrimeric G-proteins and BCR signaling.
3T3 Cells↗
The biphasic induction of p21 and p27 in breast cancer cells by modulators of cAMP is posttranscriptionally regulated and independent of the PKA pathway.
Cyclic AMP (cAMP) elevation affects growth arrest and differentiation in a wide variety of breast cell lines; however, the mechanisms associated with this process are poorly understood. Previous studies linked cAMP-mediated growth arrest in breast tumor cells to increased levels of cyclin kinase inhibitor (CKI), p21. In the present study we examined the role of cAMP-dependent protein kinase (PKA) on p21 and p27 induction in the breast cancer cell line, MDA-MB-157. The induction of the CKIs by modulators of cAMP such as cholera toxin (CT) + 1-isobutyl-3-methylxanthine (IBMX) and lovastatin fluctuates with biphasic kinetics (although the kinetics of CKI induction with CT + IBMX treatment are different from that of lovastatin) and is depicted by the periodic accumulation of lower molecular weight forms of p21 and p27 which also correlate with fluctuations in CDK2 activity. Using three different approaches we show that the cAMP-mediated induction of CKIs is independent of PKA activity. In the first approach we treated MDA-MB-157 cells with a variety of cAMP modulators such as CT + IBMX, and forskolin in the presence or absence of H-89, a potent PKA inhibitor. This analysis revealed that the cAMP activators were capable of inducing p21 even though PKA activity was completely eliminated. In the second approach PKA dominant negative stable clones of MDA-MB-157 treated with CT + IBMX or forskolin also resulted in p21 induction, in the absence of any PKA activity. Last, treatment of MDA-MB-157 cells with lovastatin, another known cAMP modulator which also causes growth arrest, resulted in the induction of p21 and p27 without any increase in PKA activity. Collectively, the above results suggest that the induction of p21 by cAMP is through a novel pathway, independent of PKA activity.
1-Methyl-3-isobutylxanthine↗
Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase.
In this paper we present the finding that lovastatin arrests cells by inhibiting the proteasome, which results in the accumulation of p21 and p27, leading to G1 arrest. Lovastatin is an inhibitor of hydroxymethyl glutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Previously, we reported that lovastatin can be used to arrest cultured cells in the G1 phase of the cell cycle, resulting in the stabilization of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27. In this report we show that this stabilization of p21 and p27 may be the result of a previously unknown function of the pro-drug, beta-lactone ring form of lovastatin to inhibit the proteasome degradation of these CKIs. The lovastatin mixture used in this study is 80% open-ring form and 20% pro-drug, beta-lactone form. We show that while the lovastatin open-ring form and pravastatin (a lovastatin analogue, 100% open ring) inhibit the HMG-CoA reductase enzyme, lovastatin pro-drug inhibits the proteasome but does not inhibit HMG-CoA reductase. In addition, many of the properties of proteasome inhibition by the pro-drug are the same as the specific proteasome inhibitor lactacystin. Lastly, mevalonate (used to rescue cells from lovastatin arrest) unexpectedly abrogates the lactacystin and lovastatin pro-drug inhibition of the proteasome. Mevalonate increases the activity of the proteasome, which results in degradation of the CKIs, allowing lovastatin- and lactacystin-arrested cells to resume cell division. The lovastatin-mediated inhibition of the proteasome suggests a unique mechanism for the chemopreventative effects of this agent seen in human cancer.
Cell Cycle↗
B cell-specific activator protein prevents two activator factors from binding to the immunoglobulin J chain promoter until the antigen-driven stages of B cell development.
The immunoglobulin J chain gene is inducibly transcribed in mature B cells upon antigen recognition and a signal from interleukin-2 (IL-2). B cell-specific activator protein (BSAP), a transcription factor that silences J chain transcription, has been identified as a nuclear target of the IL-2 signal. The levels of BSAP progressively decrease in response to IL-2 and this change correlates with the differentiation of B cells into antibody secreting plasma cells. Here we report the binding of the upstream stimulatory factor (USF) to an E-box motif immediately upstream from the BSAP site on the J chain promoter. Mutations in the USF binding motif significantly decrease J chain promoter activity in J chain expressing B cell lines. We also show that a functional relationship exists between USF and a second J chain positive-regulating factor, B-MEF2, using co-immunoprecipitation assays and transfections. Finally, we provide evidence that the binding of BSAP prevents USF and B-MEF2 from interacting with the J chain promoter during the antigen-independent stages of B cell development. It is not until the levels of BSAP decrease during the antigen-driven stages of B cell development that both USF and B-MEF2 are able to bind to their respective promoter elements and activate J chain transcription.
Animals↗
SPI-B activates transcription via a unique proline, serine, and threonine domain and exhibits DNA binding affinity differences from PU.1.
SPI-B is a B lymphocyte-specific Ets transcription factor that shares a high degree of similarity with PU.1/SPI-1. In direct contrast to PU.1(-/-) mice that die in utero and lack monocytes, neutrophils, B cells, and T cells, Spi-B-/- mice are viable and exhibit a severe B cell proliferation defect. Since PU.1 is expressed at wild type levels in Spi-B-/- B cells, the mutant mice provide genetic evidence that SPI-B and PU.1 have at least some non-redundant roles in B lymphocytes. To begin to understand the molecular basis for these defects, we delineated functional domains of SPI-B for comparison to those of PU.1. By using a heterologous co-transfection system, we identified two independent transactivation domains in the N terminus of SPI-B. Interestingly, only one of these domains (amino acids 31-61), a proline/serine/threonine-rich region, unique among Ets proteins, is necessary for transactivation of the immunoglobulin lambda light chain enhancer. This transactivation motif is in marked contrast to PU.1, which contains acidic and glutamine-rich domains. In addition, we describe a functional PU.1 site within the c-FES promoter which SPI-B fails to bind efficiently and transactivate. Finally, we show that SPI-B interacts with the PU.1 cofactors Pip, TBP, c-Jun and with lower affinity to nuclear factor interleukin-6beta and retinoblastoma. Taken together, these data suggest that SPI-B binds DNA with a different affinity for certain sites than PU.1 and harbors different transactivation domains. We conclude that SPI-B may activate unique target genes in B lymphocytes and interact with unique, although currently unidentified, cofactors.
Amino Acid Sequence↗
Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy.
Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.
Centromere↗
Linkage of chromosome 1 markers to alcoholism-related phenotypes by sib pair linkage analysis of principal components.
Using the Collaborative Study on the Genetics of Alcoholism data and affected-sib-pair linkage methods, Reich et al. [1998] reported linkage of alcohol dependence to a region near D1S1588 on chromosome 1. In this paper, we assessed the ability of multivariate sib-pair linkage analysis of the neurophysiologic measurements (including age and sex) to evaluate evidence for linkage to chromosome 1. Principal components of 16 neurophysiologic measurements, plus age and sex, were analyzed separately using sib-pair linkage analysis, and a cumulative sum of the resulting t2-statistics computed at each point on the chromosome. The first four principal components, which accounted for 74% of the total variation, showed little or no evidence for linkage in the D1S1588 region, while the remaining components showed substantial evidence for linkage. We conclude that potentially important linkage results can be missed if investigators limit attention only to major sources of variability.
Age Factors↗
Insulin-induced vasodilatation of internal carotid artery.
The increase in leg and forearm blood flow induced by insulin could be secondary to its metabolic effect on glucose uptake. We therefore investigated whether insulin causes vasodilation of the internal carotid artery, since the brain is not dependent on insulin for glucose uptake, to demonstrate that the vasodilatory effect of insulin is primary and independent of its metabolic effect. Internal carotid artery diameter was continuously monitored using a 7.5-MHz transducer linked to an Acuson XP10 ultrasonograph (Mountainview, CA) during infusion of 125 mL 10% dextrose mixed with 3 U regular insulin and 5 mmol potassium chloride over 1 hour. The internal carotid artery diameter increased progressively with time from a mean of 5.4+/-1 mm to 5.7+/-1 mm at 15 minutes, 5.9+/-1.1 mm at 30 minutes, 6+/-1.1 mm at 45 minutes, and 6.1+/-1.1 mm at 60 minutes (P < .05), an increase of 13% over baseline. Glucose was maintained between 93 and 106 mg/dL, and insulin increased from 15+/-14 microU/mL and was maintained between 34 and 47 microU/mL. There was no change in mean arterial blood pressure (MABP) or heart rate during the infusion. We conclude that insulin dilates the internal carotid artery consistently at physiological concentrations, probably independently of glucose uptake by the brain. Alterations in this effect of insulin may be of relevance in the pathogenesis of abnormalities of cerebral blood flow in type 1 and type 2 diabetics as described by our group previously.
Adult↗
The influence of patellofemoral pain on lower limb loading during gait.
OBJECTIVE: To determine if subjects with patellofemoral pain demonstrate excessive lower limb loading during gait. DESIGN: Prospective study utilizing a group of patients with patellofemoral pain and a control group. BACKGROUND: Increased rate of lower limb loading has been hypothesized as being contributory to knee osteoarthritis and may be the result of decreased knee flexion during weight acceptance. Since patients with patellofemoral pain have been reported to limit knee flexion during gait, these individuals may be at risk for the adverse effects of impulse loading. METHODS: Force plate parameters, lower extremity kinematics and stride characteristics were recorded in 15 females with patellofemoral pain and 10 pain-free controls during self-selected free and fast walking velocities. RESULTS: Individuals in the patellofemoral pain group demonstrated a significantly slower gait velocity during the free and fast trials as well as decreased stance phase knee flexion during fast walking. The average peak loading rate for the patellofemoral pain group was significantly less than the control group during both free (P=0.004) and fast walking (P=0. 03). CONCLUSIONS: Despite diminished stance phase knee flexion during fast walking, subjects with patellofemoral pain did not demonstrate increased lower limb loading. During gait, the ground reaction forces appeared to be minimized by adopting a slower walking velocity. RELEVANCE: These results indicate that altered knee kinematics as a result of patellofemoral pain do not place these individuals at risk for the adverse effects of impulse loading.
Adolescent↗
PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction.
PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.
Animals↗
Long-term survival after epirubicin, cisplatin and fluorouracil for gastric cancer: results of a randomized trial.
We report the final results of a prospectively randomized study that compared the combination of epirubicin, cisplatin and protracted venous infusion fluorouracil (5-FU) (ECF regimen) with the standard combination of 5-FU, doxorubicin and methotrexate (FAMTX) in previously untreated patients with advanced oesophagogastric cancer. Between 1992 and 1995, 274 patients with adenocarcinoma or undifferentiated carcinoma were randomized from eight oncology centres in the UK and analysed for response and survival. The overall response rate was 46% (95% confidence interval (CI), 37-55%) with ECF, and 21% (95% CI, 13-28%) with FAMTX (P = 0.00003). The median survival was 8.7 months with ECF and 6.1 months with FAMTX (P = 0.0005). The 2-year survival rates were 14% (95% CI, 8-20%) for the ECF arm, and 5% (95% CI, 2-10%) for the FAMTX arm (P = 0.03). Histologically complete surgical resection following chemotherapy was achieved in ten patients in the ECF arm (three pathological complete responses to chemotherapy) and three patients in the FAMTX arm (no pathological complete responses). The ECF regimen resulted in a response and survival advantage compared with FAMTX chemotherapy. The probability of long-term survival following surgical resection of residual disease is increased by this treatment. The high response rates seen with ECF support its use in the neoadjuvant setting.
Antineoplastic Combined Chemotherapy Protocols↗
A randomised clinical trial of antiseptic nasal carrier cream and silver nitrate cautery in the treatment of recurrent anterior epistaxis.
Sixty-four consecutive patients with a history of recurrent epistaxis were randomly assigned in the outpatient clinic to receive treatment with either Naseptin antiseptic nasal carrier cream alone (Group A) or a combination of Naseptin cream and silver nitrate cautery (Group B). Results were available on 50 patients, 22 in Group A and 28 in Group B. Twenty patients (91%) in Group A and 25 patients (89%) in Group B demonstrated improvement in their symptoms. There was no statistically significant difference in outcome between the two treatment arms (P = 0.7569). On comparing the different age groups (under and over 16 years) in the two treatment arms, once again there was no statistically significant difference in the treatment outcome (P = 1.000). In conclusion, silver nitrate cautery offers no added advantage to the management of simple epistaxis in both children and adults.
Administration, Intranasal↗
Newborn screening for human immunodeficiency virus infection in the Bronx, NY, and evolving public health policy.
New advances in the diagnosis and treatment of HIV infection continue to propel changes in public understanding of HIV infection and the administration of public health law. Over the past decade, New York State has moved from a policy of blind newborn screening for seroprevalence data to mandatory HIV testing as part of the statewide Newborn Screening (NBS) Program. A new statewide program of expedited HIV testing (48-hr turnaround results) of pregnant women and newborns (whose HIV status is unknown at the time of delivery) began in the summer of 1999. To better understand the impact this program might have on the patients who receive health care at Lincoln Medical and Mental Health Center (Lincoln Hospital), we evaluated our experience with the current NBS program prior to inauguration of the new expedited testing program. We evaluated the NBS program from February 1, 1997, to January 31, 1999, including total number of HIV-exposed/infected infants born, mother's HIV status (if known) at the time of delivery, amount of time between blood sampling and return of the test results to the Hospital, and medical follow-up of infants with positive newborn screening test results. This was a retrospective study of the NBS registry and the medical records of patients who receive primary health care from the Pediatric Immunology Service of the Department of Pediatrics at Lincoln Hospital. One hundred and four newborns were identified with positive-HIV antibody (HIV-Ab-positive), and 13 (12.5%) were confirmed to be HIV-infected by positive polymerase chain reaction (PCR) test of viral DNA. Sixty-five (62.5%) of the newborns with positive NBS screening test results were born to mothers who were known to be HIV-infected prior to delivery; 39 (37.5%) were unanticipated. Four (30%) of the 13 HIV-infected babies were born to mothers who were known to be HIV-infected prior to delivery, and 9 (70%) were born to mothers whose HIV status was unknown at the time of delivery. Eighty percent (80%) of HIV-Ab-positive infants continued to receive follow-up care at Lincoln Hospital. Relocation to other health-care facilities occurred as a result of parental choice or due to foster care placement. No babies were "lost" from the NBS program. The average time between sampling and receipt of results for all blood tests was 16 days (range 10 to 141). Nearly 40% of newborns who acquired HIV infection from their mothers were unanticipated because the mother's HIV status was unknown at the time of delivery. These unanticipated HIV-infected infants represent missed opportunities for prevention of maternal-to-child transmission of HIV infection and early therapeutic intervention for HIV-infected infants. The new expedited HIV-testing program for New York State will facilitate early diagnosis, prevention and treatment of the HIV-exposed/infected infant for whom maternal HIV status is unknown at the time of delivery.
Female↗
Intestinal obstruction following colposuspension.
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Journal Article↗
Manometric tests of anorectal function in healthy adults.
OBJECTIVE: Although tests of anorectal function are useful in the assessment of defecation disorders, there is inadequate and inconsistent information regarding normative data. Also, there are discrepancies in manometric techniques and data interpretation. Our aim was to perform a comprehensive evaluation of anorectal function in healthy adults. METHODS: We used a 6-mm diameter probe containing six radially arrayed microtransducers, and a 4-cm-long latex balloon for performing anorectal manometry in 45 healthy subjects who were controlled for gender and age. Sequentially, subjects were asked to squeeze, bear down, or blow up a party balloon. Subsequently, rectal sensation, rectal compliance, and rectoanal reflexes were assessed simultaneously by performing intermittent phasic balloon distentions. Additionally, balloon defecation, pudendal nerve latency, and saline continence tests were performed. RESULTS: In men, the anal sphincter was longer (p < 0.05) and squeeze sphincter pressure and squeeze duration were higher (p < 0.01), but resting sphincter pressure was similar to that in women. When bearing down, although not significant, the defecation index was higher in men. Distinct thresholds for rectal sensation were identified but there was no gender difference. Likewise, rectal compliance and balloon expulsion time were similar. However, during saline infusion, the onset of first leak and total volume retained were higher (p < 0.001) and pudendal nerve latency was shorter (p < 0.05) in men. Overall, parity or age did not influence anorectal function. CONCLUSIONS: This study represents the most comprehensive age- and gender-controlled assessment of anorectal function using solid state technology. Gender influences some parameters of anorectal function. Our results could serve as a valuable resource of normative data.
Action Potentials↗