Biomedical subjects
Publications and source records attributed to S Krishnan.
Stemming from work on a previous clinical candidate, loviride, and other alpha-APA derivatives, a new series of potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been synthesized. The ITU analogues, which contain a unique diarylated imidoyl thiourea, are very active in inhibiting both wild-type and clinically important mutant strains of HIV-1.
Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)
OBJECTIVE: T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells. METHODS: TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. RESULTS: DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. CONCLUSION: These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.
A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR zeta-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR zeta-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3' untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P = 0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P = 0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a -76T insertion compared to the published sequence, in all of the SLE samples and controls. RT-PCR analysis of the TCR zeta-chain 3' untranslated region showed a 344 bp product in addition to the expected 906 bp product. Cloning and sequence analysis of the 344 bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672-1233 of TCR zeta-chain mRNA. Unlike the nomal TCR zeta-chain, the expression of TCR zeta-chain with the alternatively spliced 344 bp 3' untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906 bp TCR zeta-chain 3' untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P = 0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3' untranslated region may affect the stability and/or transport of TCR zeta-chain mRNA, leading to its downregulation in SLE T cells.
The purpose of this study was to evaluate the interactive toxicity of ethanol with potassium dichromate (K2Cr2O7-chromium). Young, male Wistar rats (100-120 g) were divided into four groups of five or six animals each and were dosed, through water, with 10% ethanol (vol./vol.) or 25 ppm chromium or were dosed with a combination of ethanol+chromium at the same concentrations for a period of 22 weeks ad libitum and were maintained on normal diet. Control animals were maintained on a normal diet and water for the same period. The serum succinate dehydrogenase and liver total triglyceride levels were significantly reduced in the three treated groups. The serum alkaline phosphatase levels were significantly reduced in ethanol-treated rats, and there was no significant change in the acid phosphatase activity. Serum aspartate and alanine aminotransferase levels in the three treated groups were significantly increased. The liver glycogen significantly decreased in both the ethanol-treated and the chromium-treated rats. There was a significant increase in liver total cholesterol levels in chromium-treated rats. Total glutathione levels were significantly decreased in the livers of ethanol-treated and ethanol+chromium-treated rats. To further substantiate these findings, a histological examination of the liver and kidneys was undertaken. The livers of alcohol-treated animals showed altered hepatic architecture in the centrilobular and periportal areas, with increased sinusoidal space (space of Disse), vacuolation, and necrosis of hepatocytes. Similar changes were observed in a histological examination of the livers of chromium-treated rats, except that the damage to the hepatocytes was more confined to the periportal area. Moreover, histological examination of the livers of ethanol+chromium-treated rats revealed uniform damage in the centrilobular and periportal areas, as was observed in the groups treated either with ethanol or chromium. The histological examination of the kidneys in the three treated groups revealed significant damage to the renal tubules and Bowman's capsule, which showed vacuolation and degeneration of the basement membrane. These findings correlate well with the serum enzyme levels found in the treated groups. It is evident from this study that chronic ethanol consumption sensitizes the liver to the toxic action of agents such as chromium. It leads to impairment of the biochemical functions in the liver, and it causes liver and kidney damage. Long-term simultaneous exposure to ethanol and chromium may cause severe health problems in people who are alcoholics and work in chrome-plating and leather-tanning industries.
The precise nature and development of the memory (CD45RO+) CD4+ T lymphocytes remain unknown. In this study, we analyzed differential gene expression of human memory CD4+ T lymphocytes in relation to their naive counterparts. A suppression subtractive hybridization technique was used to isolate and clone differentially expressed genes in the memory subset with respect to the naive subset. We screened approximately 300 clones by dot blot analysis and sequenced 23 differentially expressed clones. GenBank sequence homology search showed that these clones included genes for transcription factors, enzymes and immunomodulatory molecules. Differential expression of a subset of these genes was further confirmed by RT-PCR and densitometric analysis revealed that they were expressed five to eightfold more in memory than naive CD4+ T lymphocytes. Collectively, these results suggest that multiple genes with different functions contribute to the development of immunological memory in human T lymphocytes.
Determination and interpretation of fungal gene expression profiles based on digital reconstruction of expressed sequenced tags (ESTs) are reported. A total of 51,524 DNA sequence files processed with PipeOnline resulted in 9775 single and 5660 contig unique ESTs, 31.2% of a typical fungal transcriptome. Half of the unique ESTs shared homology with genes in public databases, 35.8% of which are functionally defined and 64.2% are unclear or unknown. In Aspergillus nidulans 86% of transcripts associate with intermediate metabolism functions, mainly related to carbohydrate, amino acid, protein, and peptide biosynthesis. During asexual development, A. nidulans unexpectedly accumulates stress response and inducer-dependent transcripts in the absence of an inducer. Stress response genes in A. nidulans ESTs total 1039 transcripts, contrasting with 117 in Neurospora crassa, a 14.3-fold difference. A total of 5.6% of A. nidulans ESTs implicate inducer-dependent cell wall degradation or amino acid acquisition, 3.5-fold higher than in N. crassa. Accumulation of stress response and inducer-dependent transcripts suggests general derepression of cis-regulation during terminal asexual development.
The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.
Obstructive sleep apnoea is a complex multifactorial condition produced by a combination of anatomical and physiological factors. There is a significant associated mortality and morbidity to obstructive sleep apnoea. There is an at least 25 per cent increased mortality from cardiovascular disease when obstructive sleep apnoea patients are compared to age and gender matched healthy people. Obstructive sleep apnoea sufferers also have a much higher industrial and motor vehicle accident rate. Management of the condition should be undertaken by a multidisciplinary team including respiratory physicians, sleep laboratory technicians, otorhinolaryngologists, oral and maxillofacial surgeons and dental specialists. The diagnostic and therapeutic interactions of team members are the key to successful treatment. The treatment regime utilises nasal continuous positive airway pressure devices, mandibular advancement splints and soft and hard tissue surgery. This review provides the dental practitioner with an introduction to obstructive sleep apnoea with particular emphasis on the orofacial aspects.
Sounds generated due to rubbing of knee-joint surfaces may lead to a potential tool for noninvasive assessment of articular cartilage degeneration. In the work reported in the present paper, an attempt is made to perform computer-assisted auscultation of knee joints by auditory display (AD) of vibration signals (also known as vibroarthrographic or VAG signals) emitted during active movement of the leg. Two types of AD methods are considered: audification and sonification. In audification, the VAG signals are scaled in time and frequency using a time-frequency distribution to facilitate aural analysis. In sonification, the instantaneous mean frequency and envelope of the VAG signals are derived and used to synthesize sounds that are expected to facilitate more accurate diagnosis than the original signals by improving their aural quality. Auditory classification experiments were performed by two orthopedic surgeons with 37 VAG signals including 19 normal and 18 abnormal cases. Sensitivity values (correct detection of abnormality) of 31%, 44%, and 83%, and overall classification accuracies of 53%, 40%, and 57% were obtained with the direct playback, audification, and sonification methods, respectively. The corresponding d' scores were estimated to be 1.10. -0.36, and 0.55. The high sensitivity of the sonification method indicates that the technique could lead to improved detection of knee-joint abnormalities; however, additional work is required to improve its specificity and achieve better overall performance.
After heat treatment, human T lymphocytes downregulate the T-cell receptor (TCR)/CD3-mediated [Ca(2+)](i) response and production of inositol triphosphate. Here we demonstrate that heat treatment of T lymphocytes at sublethal temperature decreases the expression of TCR zeta chain, which plays a critical role in the regulation of TCR/CD3-mediated signal transduction. Downregulation of TCR zeta chain in heat-treated T cells was observed at 8 h and reached a maximum at 16 h. Under these conditions, the expression of CD3 epsilon or TCR alphabeta chains was minimally affected. Consistent with the decrease in TCR zeta chain, a reduction in the level of TCR/CD3 induced tyrosine phosphorylation of several cellular protein substrates, and a delay in the kinetics of peak tyrosine phosphorylation was observed in heat-treated T cells. Interestingly, analysis of the TCR zeta chain content in the detergent-insoluble membrane fraction showed that heat treatment induces translocation of soluble TCR zeta chain to the cell membranes. In addition, the mRNA level of TCR zeta chain was reduced in heat-treated T cells. Correlative with the downregulation of TCR zeta chain mRNA, the level of the TCR zeta chain transcription factor Elf-1 was also reduced in heat-treated cells. We conclude that heat stress causes a decrease in the level of TCR zeta chain by increasing its association with the membranes and decreasing the transcription of the TCR zeta gene. Decreased expression of the TCR zeta chain is apparently responsible for the decreased TCR/CD3 responses of T cells.
Phthalate esters have been implicated as xenoestrogens. One among them is di-ethylphthalate (DEP), which is used as plasticizer, detergent base, and binder in incense sticks and after-shave lotions. DEP is one of the contaminants of freshwater and marine ecosystems. Incense stick workers are occupationally exposed to DEP and some workers are chronic alcoholics. Therefore, a study was undertaken to evaluate the interactive toxicity of DEP with ethyl alcohol (EtOH) in young male Sprague-Dawley rats. The rats were given 50 ppm DEP (w/v), 5% EtOH (v/v) and a combined dose of 50 ppm DEP (w/v)+EtOH (5% v/v) in water ad libitum for a period of 120 days and were maintained on normal diet. Control animals received normal diet and plain water. During the treatment rats were weighed every week and water consumption per day was measured. After the completion of treatment, liver weight/body weight, liver weight, body weight, serum enzymes and other biochemical parameters were assessed. It was found that there was no significant change observed in body weight, liver weight, liver weight/body weight and water consumption. It was observed that there was a significant decrease in liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in EtOH, DEP and EtOH+DEP treated rats in the order of EtOH>DEP>EtOH+DEP as compared with control. Serum AST, ALT, acid phosphatase (ACP), alkaline phosphatase (ALP), succinate dehydrogenase (SDH) and liver ACP showed significant increase in DEP and EtOH+DEP treated rats in the order of DEP>EtOH+DEP as compared with control and EtOH treated rats. On the contrary, there was no significant change in liver ALP levels in treated rats. There was significant increase in liver SDH, glycogen, total triglyceride, total cholesterol and lipid peroxidation in DEP and EtOH+DEP treated rats, but no significant changes in the serum SDH, glucose and total triglyceride levels. Serum total cholesterol levels in DEP and EtOH+DEP treated rats were significantly high as compared to control and EtOH treated rats. These results show that there is no interaction of DEP with EtOH but DEP alone leads to severe impairment of lipid metabolism coupled with toxic injury to the liver as evident from significantly altered lipid and enzyme levels in the liver and serum. Long term simultaneous exposure to DEP and EtOH may have severe implications for humans who are occupationally exposed to these two xenobiotics.
The total structure factor S(Q) and the radial distribution function G(r) of liquid Y3Al5O12 (YAG) were measured at 1770-2230 K by x-ray scattering from samples under containerless conditions in Ar and O2. Nominal coordination numbers are 4 for Al3+ and 6 for Y3+ ions. The G(r) has peaks at r approximately 1.8 A for Al-O, r approximately 2. 25 A for Y-O, and r approximately 3.3-3.6 A assigned to metal ions in adjacent AlO5-4 and YO9-6 polyhedral ions. Relative to the pure oxides, G(r) for molten YAG has smaller half-widths for the Al-O and Y-O peaks, and an increased sensitivity to temperature and the ambient gas composition.
Previous studies have determined that the frequency of germ-line p53 mutations in familial breast cancer patients is 1% or less, but these reports have not investigated the importance of polymorphic intron base changes in the p53 gene. Therefore, we investigated the frequency of both exon and intron germ-line p53 base changes in 42 breast cancer patients with a strong family history of breast cancer. The mean age of presentation of these patients was 44.0 years (range, 29-69), and 12 of 42 (29%) were of known Ashkenazi ancestry. Purified DNA obtained from the 42 index cases was screened for germ-line p53 mutations in exons 2-11 and surrounding introns using a combination of intron based primers for PCR-single strand conformation polymorphism analysis, direct sequencing, and microarray sequencing using the Affymetrix p53 gene chip methodology. Morphological analysis of apoptosis and cell survival determination were performed on EBV-immortalized lymphoblastoid cell lines from two patients with the p53 intron 6 mutation. A germ-line mutation in the p53 gene at nucleotide 13964 with a G to C base change (13964GC) was identified in 3 of 42 (7.1%) hereditary breast cancer patients. Two patients were heterozygous for this mutation, and one patient had a homozygous mutation. In comparison, 0 of 171 (0%) of sporadic breast cancer patients had the p53 13964GC mutation (P = 0.0003). We found that 0 of 42 (0%) of these hereditary breast cancer patients had other germ-line p53 mutation in exons 2-11. However, pedigree analysis demonstrated that all three patients had strong family histories of multiple types of cancers consistent with Li-Fraumeni syndrome but with late age of onset. Comprehensive BRCA1 and BRCA2 nucleotide analysis from patients with the p53 13964GC mutation revealed no concomitant deleterious BRCA1 or BRCA2 mutations, although they were found in the other hereditary breast cancer patients. Functional analysis of two immortalized lymphoblastoid cell lines derived from patients with the p53 13964GC mutation demonstrated prolonged in vitro survival in response to cisplatinum treatment and showed decreased chemotherapy-induced apoptosis. Immunohistochemical analysis of breast tumors from these patients revealed high levels of mutant p53 protein, suggesting a functional mutation in the p53 gene. In summary, we have identified a single p53 intron mutation in familial breast cancer patients that is present at elevated frequency and has functional activity.
A novel de-noising method for improving the signal-to-noise ratio of knee-joint vibration signals (also known as vibro-arthrographic (VAG) signals) is proposed. The de-noising methods considered are based on signal decomposition techniques, such as wavelets, wavelet packets and the matching pursuit (MP) method. Performance evaluation with synthetic signals simulated with the characteristics expected of VAG signals indicates good de-noising results with the MP method. Statistical pattern classification of non-stationary signal features extracted from time-frequency distributions of 37 (19 normal and 18 abnormal) MP method-de-noised VAG signals shows a sensitivity of 83.3%, a specificity of 84.2% and an overall accuracy of 83.8%.
Explore the source record for details and available documents.
Vibroarthrographic (VAG) signals emitted by human knee joints are nonstationary and multicomponent in nature; time-frequency distributions (TFD's) provide powerful means to analyze such signals. The objective of this paper is to construct adaptive TFD's of VAG signals suitable for feature extraction. An adaptive TFD was constructed by minimum cross-entropy optimization of the TFD obtained by the matching pursuit decomposition algorithm. Parameters of VAG signals such as energy, energy spread, frequency, and frequency spread were extracted from their adaptive TFD's. The parameters carry information about the combined TF dynamics of the signals. The mean and standard deviation of the parameters were computed, and each VAG signal was represented by a set of just six features. Statistical pattern classification experiments based on logistic regression analysis of the parameters showed an overall normal/abnormal screening accuracy of 68.9% with 90 VAG signals (51 normals and 39 abnormals), and a higher accuracy of 77.5% with a database of 71 signals with 51 normals and 20 abnormals of a specific type of patellofemoral disorder. The proposed method of VAG signal analysis is independent of joint angle and clinical information, and shows good potential for noninvasive diagnosis and monitoring of patellofemoral disorders such as chondromalacia patella.
BACKGROUND/AIMS: Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice. METHODS: Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [(14)C]butyrate or glucose, and production of (14)CO(2), as well as of intermediate metabolites (acetoacetate, beta-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined. RESULTS: Butyrate oxidation (micromol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (micromol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of beta-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread. CONCLUSIONS: Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation.