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Biomedical subjects

A Mittal

Publications and source records attributed to A Mittal.

At least 19 recordsLinked to original sources
PubMed2004

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Amino Acid Sequence↗
PubMed2004

Matched study of three methods for palliation of malignant pyloroduodenal obstruction.

BACKGROUND: The traditional management of pyloroduodenal obstruction is open gastrojejunostomy (OGJ). More recently laparoscopic gastrojejunostomy (LGJ) and endoscopic stenting (ES) have been introduced. The aim of this study was to evaluate the three approaches to the palliation of malignant pyloroduodenal obstruction. METHODS: All patients who underwent surgery (open and laparoscopic) for malignant pyloroduodenal obstruction at Auckland City Hospital between 1989 and 2002 inclusive were identified from International Classification of Diseases (ICD) 10 codes and from the Otago Surgical Audit Database. Patients who had an endoscopic stent were identified from the Endoscribe database. A review of medical records was conducted and data recorded in a structured pro forma. There were 181 patients with malignant pyloroduodenal obstruction of whom 56 patients had OGJ, 14 had LGJ and 16 had ES. Patients in the LGJ and ES groups were matched with those who underwent OGJ with respect to American Society of Anesthesiologists (ASA) grade (I-V), age (within 10 years) and level of obstruction (pylorus, first part of duodenum D1, D2, D3 and D4). The primary outcomes compared between the groups were time to starting free oral fluids and light diet, length of stay and survival. RESULTS: There were no significant differences in age, sex, ASA grade and level of obstruction between the matched OGJ (n=16), LGJ (n=14) and ES (n=16) groups. There was a significant reduction in time to starting free oral fluids and light diet, and length of stay after the procedure, in the ES group. Patients who underwent surgical palliation of the obstruction had significantly more complications than those who underwent stenting (P=0.016). There were no significant differences in requirement for biliary drainage either before or after the procedure between the three groups. Survival was shortest in the ES group. CONCLUSION: This matched study showed significant advantages for ES compared with OGJ and LGJ in the palliation of malignant pyloroduodenal obstruction.

Adult↗
PubMed2004

Mitochondrial involvement in RP-1 mediated apoptosis in U 87 cells.

The aqueous extract of RP-1, which rendered significant protection to whole body irradiated mice, was found to be tumoricidal. The mode of cytotoxic action of RP-1 attributing to its antitumor action was investigated in U 87 cells with special reference to mitochondrial contribution. RP-1 doses above 0.5 microg/ml reduced colonogenic survival (maximum reduction of 62% at 10 microg/ml) and increased the free radical generation, G2/M fraction and apoptotic frequency. Prolonged exposure to RP-1 rendered significant increase in mitochondrial mass. It also reduced mitochondrial membrane potential in a dose and time dependent manner that was restored by verapamil, a Ca+2 channel blocker. Mitochondrial anti-apoptotic proteins Bcl-2 and Hsp-70 levels were also reduced by RP-1 treatment in a dose and time dependent manner. The ability of RP-1 to disrupt mitochondrial structure and function could be responsible for its cytotoxic action.

Antineoplastic Agents↗
PubMed2004

Cytokine expression pattern in the genital tract of Chlamydia trachomatis positive infertile women - implication for T-cell responses.

Human genital infection caused by Chlamydia trachomatis is thought to be immunologically mediated, resulting in local recruitment of lymphocyte subsets and inducing the production of cytokines. Little information is available about the role of lymphocyte recruitment and the regulation of cytokine production in the genital tract of C. trachomatis positive infertile women. We have evaluated the recruitment of lymphocyte subsets in the genital tract and production of Th1/Th2 cytokines in cervical secretions and laparoscopic specimens from the fallopian tubes of C. trachomatis positive infertile women (n = 17) and compared them with controls, viz. C. trachomatis negative infertile women (n = 20) using ELISA and flow cytometry. None of these patients were found to be infected either with Candida sps., bacterial vaginosis, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasma hominis or Ureaplasma urealyticum in the cervix. Flow cytometric analysis of cervical secretions in Chlamydia positive women revealed recruitment of both CD4 and CD8 lymphocytes to the genital tract was up-regulated and a variation in the production rates of different cytokines in cervical secretions and fallopian tube was observed. We found that the immune responses in cervical secretions were of Th0 type, since all the analysed cytokines, viz. IFN-gamma, TNF-alpha, IL-10 and IL-12 were up-regulated. As, both CD4 and CD8 cells contribute to the production of IFN-gamma and IL-10, these results suggest that along with CD4 cells, CD8 lymphocytes also may be important for local regulation of Th1/Th2 responses in the genital tract during C. trachomatis infection.

Adult↗
PubMed2004

Protein structure and fold prediction using tree-augmented naive Bayesian classifier.

For determining the structure class and fold class of Protein Structure, computer-based techniques have became essential considering the large volume of the data. Several techniques based on sequence similarity. Neural Networks, SVMs, etc have been applied. This paper presents a framework using the Tree-Augmented Networks (TAN) based on the theory of learning Bayesian networks but with less restrictive assumptions than the naive Bayesian networks. In order to enhance TAN's performance, pre-processing of data is done by feature discretization and post-processing is done by using Mean Probability Voting (MPV) scheme. The advantage of using Bayesian approach over other learning methods is that the network structure is intuitive. In addition, one can read off the TAN structure probabilities to determine the significance of each feature (say, Hydrophobicity) for each class, which help to further understand the mystery of protein structure. Experimental results and comparison with other works over two databases show the effectiveness of our TAN based framework. The idea is implemented as the BAYESPROT web server and it is available at http://www-appn.comp.nus.edu.sg/-bioinfo/bayesprot/Default.htm.

Artificial Intelligence↗
PubMed2003

Effect of treatment for Chlamydia trachomatis during pregnancy.

OBJECTIVES: To screen and treat chlamydial infection in pregnant women in order to assess the effects of therapeutic intervention on the outcome of Chlamydia trachomatis-infected pregnancy. METHODS: Enrolled in the study were 350 women in the first to third trimesters of pregnancy. Endocervical swabs were collected for C. trachomatis diagnosis by DFA and PCR. A few STD infections, viz.: Neisseria gonorrhoeae, Candida spp., bacterial vaginosis, Trichomonas vaginalis and Treponema pallidum were ruled out. After excluding 22 patients infected with other sexually transmitted infections, a cohort of 328 pregnant women comprised the study population. Anti-chlamydial treatment (viz.: oral therapy with erythromycin stearate, 500 mg 4 times daily for 7 days) was given to 17 women (group I) and their partners. Fifteen patients of group I were retested by DFA and PCR assay for C. trachomatis infection 2 weeks following therapy. Other Chlamydia-positive patients (n=42) who were lost to follow-up were classified as untreated positive cases (group II) while group III included C. trachomatis negative cases (n=269). Data on obstetric outcome were recorded in a total of 164 cases. Statistical comparison of the data were done using the chi(2)-test and means were compared using Student's t-test. RESULTS: Among the 350 pregnant women enrolled initially for the study, C. trachomatis positivity was found to be 18.8% (n=66) in the endocervix by DFA and PCR assay. Co-infection with Candida spp., bacterial vaginosis, T. vaginalis and T. pallidum was 2.0%, 1.7%, 1.7% and 0.5%, respectively. None of the pregnant women was infected with N. gonorrhoeae. Pregnant women at an increased risk of chlamydial infection included those who had multiple births and were in second trimester of pregnancy. Fifteen patients of group I became Chlamydia-negative following treatment. Data on obstetric outcome were recorded in 11, 26 and 127 patients of groups I, II and III, respectively. The mean duration of gestation for premature deliveries was found to be significantly higher in group I in comparison with group II [35.5 vs. 33.1 weeks (P<0.05)], thereby showing an improved effect of treatment on pregnancy outcome. The mean of low birth weight births was higher in group I compared with group II (2200.0 vs. 2113.3 g), however, this was statistically non-significant. Stillbirths were significantly higher among group II in comparison with group III [11.5% vs. 4.7% (chi(2)=1.79; P<0.5)]. No stillbirths were recorded in patients who had taken anti-chlamydial treatment. CONCLUSIONS: Our findings suggest that routine screening and treatment of C. trachomatis infection in pregnant women, especially those in high risk groups, should be mandatory to reduce the adverse effects on obstetric outcome.

Adult↗
PubMed2003

Predominance of Chlamydia trachomatis serovars associated with urogenital infections in females in New Delhi, India.

Screening for Chlamydia trachomatis was done for 280 endocervical swab samples by PCR specific for endogenous plasmid. Age dependency was seen in symptomatic patients, with a high chlamydial prevalence rate (28%) found in younger women. Genotyping by restriction fragment length polymorphism analysis of omp1 PCR-positive samples showed serovars D, E, and F to be the most prevalent.

Adolescent↗
PubMed2001

Trauma and co-morbidity--a pilot study.

AIMS: To study the adult trauma patient population at the Auckland Hospital in order to determine the age distribution of trauma, the prevalence and importance of co-morbid conditions and any effect of the latter on the length of stay in the hospital. METHODS: Data were collected on 78 consecutive patients admitted to the Auckland Hospital under the Trauma team between December 1999 and January 2000. Data were collected by interviewing the patient, as well as reviewing patient's medical notes and the Trauma Registry. RESULTS: The prevalence of co-morbidities was 14.7%. No co-morbidity was found below the age of 40 years, but the prevalence of co-morbidity increased with age. The average length of stay for patients with no comorbidities and an Injury Severity Score (ISS) >15 was 19 days while for those with co-morbidities was 24.5 days--an increase of 29%. CONCLUSION: This pilot study has found that a significant number of trauma patients being admitted to Auckland Hospital have a pre-existing co-morbid condition that may alter their length of stay. It is an important issue that warrants further investigation, in order to devise a more accurate prognostic scoring system.

Adolescent↗
PubMed2001

Comprehensive kinetic analysis of influenza hemagglutinin-mediated membrane fusion: role of sialate binding.

The data of Danieli et al. (J. Cell Biol. 133:559-569, 1996) and Blumenthal et al. (J. Cell Biol. 135:63-71, 1996) for fusion between hemagglutinin (HA)-expressing cells and fluorescently labeled erythrocytes has been analyzed using a recently published comprehensive mass action kinetic model for HA-mediated fusion. This model includes the measurable steps in the fusion process, i.e., first pore formation, lipid mixing, and content mixing of aqueous fluorescent markers. It contains two core parameters of the fusion site architecture. The first is the minimum number of aggregated HAs needed to sustain subsequent fusion intermediates. The second is the minimal number of those HAs within the fusogenic aggregate that must undergo a slow "essential" conformational change needed to initiate bilayer destabilization. Because the kinetic model has several parameters, each data set was exhaustively fitted to obtain all best fits. Although each of the data sets required particular parameter ranges for best fits, a consensus subset of these parameter ranges could fit all of the data. Thus, this comprehensive model subsumes the available mass action kinetic data for the fusion of HA-expressing cells with erythrocytes, despite the differences in assays and experimental design, which necessitated transforming fluorescence dequenching intensities to equivalent cumulative waiting time distributions. We find that HAs bound to sialates on glycophorin can participate in fusion as members of the fusogenic aggregate, but they cannot undergo the essential conformational change that initiates bilayer destabilization, thus solving a long-standing debate. Also, the similarity in rate constants for lipid mixing and content mixing found here for HA-mediated fusion and by Lee and Lentz (Proc. Natl. Acad. Sci. U.S.A. 95:9274-9279, 1998) for PEG-induced fusion of phosphatidylcholine liposomes supports the idea that subsequent to stable fusion pore formation, the evolution of fusion intermediates is determined more by the lipids than by the proteins.

Calibration↗
PubMed2001

Influence of Podophyllum hexandrum on endogenous antioxidant defence system in mice: possible role in radioprotection.

Podophyllum hexandrum, a Himalayan herb with known radioprotective and anti-tumour properties, was investigated for its mechanism of action. Glutathione S-transferase (GST), catalase, superoxide dismutase (SOD) activities and lipid peroxidation (LPx) were determined in the liver, jejunum and ileum at various time intervals, with and without the aqueous extract of P. hexandrum rhizome (200 mg/kg b.w. i.p.) in unirradiated and whole body irradiated (10 Gy,-2 h) male Swiss albino mice. Pre-irradiation treatment with P. hexandrum enhanced liver GST (P<0.01) and SOD (P<0.05) at 12 h post irradiation, the intestinal SOD (P<0.00005) at 84 h post irradiation was significantly elevated. However, no significant change was manifested in the catalase activity in the liver, at any of the post irradiation intervals (0, 12 and 84 h). The antioxidant defence with Podophyllum sp. treatment in mice can explain to some extent its protective action manifested in terms of survival against whole body lethal irradiation. However, some other possible mechanisms that may strengthen radioprotective action of the Podophyllum sp. extract need to be investigated further.

Animals↗
PubMed2000

Deployment of membrane fusion protein domains during fusion.

It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion proteins SV5 F1 and HIV gp120/41, and for the intracellular SNARE fusion system. By comparing these domains, we have constructed a minimal set which appears to be adequate to explain how the conformational changes can produce a successful fusion event, i.e. communication of aqueous compartments.

Animals↗
PubMed2000

Reduced food consumption increases water intake and modulates renal aquaporin-1 and -2 expression in autoimmune prone mice.

Aquaporin-1(AQP1) and AQP2 are members of the aquaporin family of cell membrane water channel transport proteins and have been implicated in the regulation of renal water excretion. We have previously shown that calorie restriction (CR) relative to ad libitum (AL) feeding extends lifespan and delays the onset of autoimmune kidney disease in lupus-prone (NZBxNZW)F1 (B/W) mice. To determine if AQP1 and/or AQP2 expression is influenced by CR, mice were fed an AL or CR (40% less food) diet until 4 (young) or 9 (old) months of age when mice were sacrificed. Kidneys were removed and the expression of AQP1 and AQP2 was determined at the protein and mRNA levels using western blotting and RT-PCR respectively. While age did not significantly increase AQP1 expression in the AL groups, CR did increase both the protein (1.4-fold) and mRNA (2.4-fold) levels. In old mice, AQP1 expression was higher (1.8-fold) in CR compared to the AL group while CR had no effect in young mice. In contrast, AQP2 showed an age related decrease (55%) in the AL groups and an increase in the protein (8.4-fold) and mRNA (1.7-fold) levels in the CR groups. Relative to AL, CR decreased AQP2 expression at the protein (90%) and mRNA (50%) levels in the young mice while an increase at the protein (2.9-fold) and mRNA (1.9-fold) levels was evident in the old mice. Interestingly, a significant increase in water intake per gram body weight was found in both young and old CR fed mice when compared to their AL counterparts which may contribute to the prevention of autoimmune disease with age and differences in longevity. These data show, for the first time, significant age and diet influences in renal AQP1 and AQP2 expression at both protein and mRNA levels in lupus-prone mice.

Age Factors↗
PubMed2000

Chlamydia trachomatis and human papillomavirus infection in Indian women with sexually transmitted diseases and cervical precancerous and cancerous lesions.

OBJECTIVES: Sexually transmitted diseases (STDs) and anogenital cancers are the major health problems in Indian women but no reliable estimate of the prevalence of either genital chlamydial infection or human papillomavirus (HPV) infection in STD patients is available. The aim of this study was to detect the frequency of Chlamydia trachomatis and the most prevalent high-risk HPV type 16 (HPV 16) infection in Indian women, with STDs and precancerous and cancerous lesions of the uterine cervix by polymerase chain reaction (PCR), and their comparison with those of conventional serology and antigen tests used for C. trachomatis detection. METHODS: Endocervical swabs or scrapes were collected from 50 women with STDs and 30 normal healthy women attending the STD clinics of Smt. Sucheta Kripalani Hospital, New Delhi. Scraped cervical cell specimens were also collected from 50 women with precancerous and cancerous lesions of the uterine cervix. Detection of C. trachomatis and HPV was carried out by PCR using chlamydia and HPV genome-specific oligonucleotide primers. The detection of chlamydial antigen and IgG-specific antibodies was carried out by enzyme immunoassay (EIA) and serological enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: A chlamydia plasmid-based PCR assay detected 50% (25 of 50) positivity of C. trachomatis in STD patients and HPV 16 DNA was found in 30% (15 of 50) of these cases which are significantly higher than those found in healthy controls. The PCR estimate of chlamydia was found to be higher than its reported frequency by tissue culture. The EIA could detect chlamydial antigen in only 13 cases (26%) while serological ELISA revealed evidence of chlamydia IgG-specific antibodies in 26 (52%) cases. Interestingly, in women with precancerous and cancerous lesions, the rate of HPV 16 infection was very high (52% and 72%, respectively), whereas the frequency of chlamydia infection was found to be 12-22% only. Occurrence of other sexually transmitted agents was also evaluated in the women. CONCLUSIONS: This is the first PCR estimate of genital chlamydial (50%) and HPV 16 (30%) infection in STD patients and women with precancerous and cancerous lesions of the uterine cervix in India. The PCR method seems to be a good alternative to tissue culture.

Adolescent↗
PubMed2000

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion.

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.

Animals↗
PubMed2000

Detection of Chlamydia trachomatis antigen in spontaneous abortions. Is this organism a primary or secondary indicator of risk?

In order to investigate whether or not Chlamydia trachomatis infection is a risk factor for pregnancy loss, 77 spontaneous abortion patients (6-24 weeks gestation), admitted to gynaecology emergency of Safdarjang Hospital, New Delhi, India. Twenty-five pregnant women (6-16 weeks gestation) attending the same hospital for induced abortion, were included in the study. C. trachomatis antigen was detected in endometrial curretage tissue by enzyme immunoassay (EIA). The detection rate was 15.6% (12/77) among spontaneous abortion patients and 4% (1/25) among women undergoing induced abortion. There was no statistically significant association between the mean age/mean gestational age of those experiencing spontaneous abortion, with and without C. trachomatis infection (26.9 years versus 25.06 years and 11.1 weeks versus 9.6 weeks, respectively). High prevalence of C. trachomatis was found in multigravidae and parous spontaneous abortion patients, compared with that in primigravidae and nulliparous Chlamydia-negative spontaneous aborters (75.0% versus 25.0%; 66.7% versus 33.3%, respectively). The prevalence of chlamydial antigen in patients with no prior history of spontaneous abortion was 16.1% (10/62) compared with 18.1% (2/11) in women with one prior abortion. Further study is required to determine whether C. trachomatis infection is a primary or secondary indicator of risk.

Abortion, Spontaneous↗