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Biomedical subjects

B Mittal

Publications and source records attributed to B Mittal.

At least 37 records · Page 2Linked to original sources
PubMed2000

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.

Actinin↗
PubMed1999

Radiation therapy with concomitant hydroxyurea and fluorouracil in stage II and III head and neck cancer.

PURPOSE: In 1986, a multi-institutional phase II trial was begun to study the use of chemotherapy with concomitant radiation in patients with stage II and III head and neck cancer. End points were overall survival, progression-free survival, local/regional control, and toxicity in the setting of organ preservation with concomitant treatment. METHODS: Eligible patients with stage II or III disease received chemotherapy and radiation on a 2-week cycle. Chemotherapy consisted of continuous infusion fluorouracil (5-FU) at 800 mg/m2/d for 5 consecutive days (days 1 to 5) and hydroxyurea (HU) at 1 g orally every 12 hours for 13 doses starting the evening before the start of irradiation. Radiation therapy was given as single 1.8- to 2.0-Gy fractions for 5 consecutive days (days 1 to 5) with chemotherapy. Each 5 days of treatment was followed by a 9-day break (days 6 to 14), during which no additional treatment was given. Treatment cycles were repeated until the completion of the planned radiation dose (six to eight cycles). RESULTS: Between 1989 and 1996, 60 patients were enrolled. All patients were eligible for analysis, with a median follow-up of 52 months for surviving patients and 42 months for all patients. Grade 3 to 4 mucositis occurred in 57% of patients. The 5 year-actuarial overall survival, progression-free survival, and local/regional control were 65%, 82%, and 86%, respectively. Eight patients developed local and/or regional recurrence after treatment. Surgical salvage was possible in three of these patients. Thus, the ultimate 5-year local/ regional control was 91%. CONCLUSION: Concomitant radiation and chemotherapy with 5-FU and HU is an effective regimen in patients with stage II and III head and neck cancer. Progression-free survival and local/regional control appear to be superior to expected rates in patients treated with surgery and radiation. Further testing of this regimen in a phase III setting is indicated.

Administration, Oral↗
PubMed1998

Induction chemotherapy followed by concurrent chemoradiation for advanced head and neck cancer: improved disease control and survival.

PURPOSE: To determine tumor response rate, patterns of failure, toxicity, and survival in advanced squamous head and neck cancer after a combined treatment program that consists of induction chemotherapy, organ-sparing surgery, and concurrent chemoradiation. Long-term outcome data are presented. PATIENTS AND METHODS: Between July 1991 and March 1993, 93 patients received three cycles of induction chemotherapy that consisted of cisplatin, fluorouracil (5-FU), l-leucovorin, and alpha-interferon2b (PFLl-alpha) followed by optional limited surgery and six to eight cycles of 5-FU, hydroxyurea, and concurrent radiation (FHX) to a total radiation dose of 65 to 75 Gy. RESULTS: Ninety-three patients were entered onto this study and 97% had stage IV disease, with 66 patients who were N2 or N3. Sixty-one patients (66%) achieved a clinical complete remission (CR) after induction therapy. Thirty-four patients underwent surgery. Seventy-nine patients proceeded to FHX. With a median follow-up time of 43 months for surviving patients, 20 patients have had disease progression (13 local, two distant, five both), and there have been 35 deaths (18 from disease, six treatment-related, two from a second primary, and nine for other medical reasons). At 5 years, progression-free survival is 68%, and overall survival is 62%. Surgery was organ-preserving, as only a single laryngectomy and no glossectomies were performed in primary management. Acute toxicity related to PFLl-alpha consisted of severe or life-threatening mucositis in 57% and leucopenia in 65% of patients. During FHX, 81% of patients had grade 3 or 4 mucositis. CONCLUSION: PFLl-alpha is a highly active regimen that induced clinical CR in two thirds of patients. When followed by limited surgery and FHX, resultant local and distant disease control, organ preservation, and overall 5-year survival are very promising in high-risk stage IV patients. Based on these local control and survival data, further evaluation of this treatment sequence, induction chemotherapy followed by concurrent chemoradiation, is warranted. Identification of similarly active but less toxic regimens is a high priority.

Adolescent↗
PubMed1997

Proportion and pattern of dystrophin gene deletions in north Indian Duchenne and Becker muscular dystrophy patients.

Population-based variations in frequency and distribution of dystrophin gene deletions have been recognized in Duchenne/Becker (DMD/BMD) muscular dystrophy patients. In the present study, DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization. A total of 88 (73%) patients showed intragenic deletions in the dystrophin gene. The observed proportion of gene deletions is relatively high, particularly compared with that of Asian counterparts. However, the distribution of breakpoints across the gene does not show significant variations.

Dystrophin↗
PubMed1997

An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.

Amino Acid Sequence↗
PubMed1997

Zeugmatin is part of the Z-band targeting region of titin.

Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

Animals↗
PubMed1997

The health of the health care industry: a report card from American consumers.

This article reports on the public perceptions of the quality of health care services offered by three categories of provider professionals (physicians, dentists, and pharmacists) and three categories of hospitals (public, private, and those with religious affiliations). The authors examine quality along two dimensions: the quality of "cure" vs. the quality of "caring." Results show that, although the health care industry does a fairly decent job in delivering quality service, there is uneven performance across different providers. Data also show the quality of caring to be lagging the quality of cure; importantly, however, the former is more influential in earning patient loyalty. The role of the two types of quality in patient retention are presented, along with implications for health care industry managers.

Attitude to Health↗
PubMed1997

Genotype-phenotype correlation in Duchenne/Becker muscular dystrophy patients seen at Lucknow.

The molecular basis of two allelic forms of muscular dystrophy, Duchenne (DMD) and Becker (BMD), has been explained by frame shift hypothesis. In order to test this hypothesis, deletional mutations in 59 patients confirmed to have DMD and 11 BMD patients were analysed using multiplex polymerase chain reaction and Southern hybridization with dystrophin cDNA probes. Translational reading frame of the dystrophin gene was derived from 'Border type' analysis of exons flanking the intragenic deletions. The correlation between genotype (reading frame) and phenotype (clinical severity) showed higher number of DMD patients (approximately 20%) deviating from the frame shift hypothesis. The patients who deviated had deletions at the central hot spot region of the dystrophin gene. The presence of these deviations in a large number of DMD patients highlights the difficulties in predicting the clinical progression of the disease based only on DNA profile.

Gene Deletion↗
PubMed1996

Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin.

Zeugmatin is a muscle specific protein discovered by Maher et al. [1985: J. Cell Biol. 101:1871-1883] to be in Z-Bands of muscle and in the dense bodies of smooth muscle. Maher et al. [1985] generated a zeugmatin specific monoclonal antibody, McAb20, and then used immunoaffinity chromatography to isolate a 600-800 kD protein. During myofibrillogenesis of embryonic cardiac muscle, zeugmatin is detected in fully formed Z-bands in the mature myofibrils but not in the Z-bodies of premyofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24]. Rhee et al. [1994] have postulated that zeugmatin may be responsible for the fusion of the alpha-actinin containing Z-bodies to form the solid Z-Bands of the mature myofibrils. The current studies were undertaken to characterize the properties of zeugmatin. The McAb20 was used to probe a chicken heart lamba gt11 expression library, and three unique positive clones of 1.1, 1.4, and 1.7 kB were isolated. These were inserted into pcDNA3, sequenced, and assembled into a 1.8 kB ORF. A 60% identity with N-terminal region of the human cardiac titin sequence was revealed at the amino acid level. This region of the 1.8 kB zeugmatin sequence is located entirely in the Z-band region of the human cardiac titin molecule. The 1.1 kB clone of zeugmatin was subcloned into pTrcHisC and expressed in bacteria. Bacterial lysates were prepared and run over nickel columns to isolate a 46 kD fusion protein. This fusion protein formed a complex with purified alpha-actinin that could be immunoprecipitated with the zeugmatin specific antibody, McAb 20. The 1.1 kB sequence was transfected into non-muscle cell lines, PtK2 and REF. Twenty-four hours after transfection, the 46 kD zeugmatin peptide, not present in control non-muscle cells, was localized in focal adhesions and in a punctate pattern along the stress fibers. Double immunofluorescence staining revealed that zeugmatin colocalized with the alpha-actinin in the dense bodies and focal contacts of the stress fibers. At longer time points, as the transfected cells accumulated more truncated zeugmatin molecules, the cells lost adhesion plaques and stress fibers, and became detached from the substratum. Our results indicate the zeugmatin is part of the titin molecule that is located within the Z-band and that this section of the titin molecule anchors the actin crosslinking alpha-actinin molecules.

Actinin↗
PubMed1996

The myosin filament XV assembly: contributions of 195 residue segments of the myosin rod and the eight C-terminal residues.

A mixture of two peptides of approximately M(r) 13000 has been isolated from a papain digest of LC2 deficient myosin. The peptides assemble into highly ordered aggregates which in one view are made up of strands of pairs of dots with an average side to side spacing of 13.0 nm and an average axial repeat of 9.0 nm. In another view there are strands of single dots with a side-to-side spacing of 7.8 nm and an axial repeat of 9.1 nm. From N-terminal peptide sequencing, the two peptides have been shown to come from regions of the myosin rod displaced by 195 residues. We have shown that either peptide alone can assemble to form the same aggregates. The 195 residue displacement of the M(r) 13000 peptides corresponds closely to the 196 residue repeat of charges along the myosin rod. This finding permits us to designate 195 residue segments of the myosin rod and to relate assembly characteristics directly to the similar 195 residue segments and 196 residue charge repeat. The most C-terminal 195 residue segment carries information for assembly into helical strands. The contiguous 195 residue segment, in major part, carries information for the unipolar assembly, characteristic of the assembly in each half of the myosin filament. The next contiguous 195 residue segment, in major part, carries information for bipolar assembly which is characteristic of the bare zone region of the filament; and for the transition from the bipolar bare zone to unipolar assembly. The effect of the eight C-terminal residues of the myosin rod on the assembly of the contiguous 195 residues has also been studied. The entire fragment of 195 + eight C-terminal residues assembled to form helical strands with an axial repeat of 30 nm. Successive deletion of charged residues changed the axial repeat of the helical strands suggesting that the charged residues at the C-terminus are involved in determining the pitch in the helical assembly of the contiguous 195 residues.

Amino Acid Sequence↗
PubMed1996

Alcohol inhibits cell-cell adhesion mediated by human L1.

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector-transfected controls. Ethanol potently and completely inhibited L1-mediated adhesion both in transfected L cells and NIH/3T3 cells. Half-maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n-butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1-mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N-CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.

3T3 Cells↗
PubMed1996

High frequency of new mutations in North Indian Duchenne/Becker muscular dystrophy patients.

Accurate carrier determination is an important aspect in providing prenatal diagnosis and genetic counselling to families with Duchenne/Becker muscular dystrophy patients. Using quantitative polymerase chain reaction, we have analyzed the carrier status of 31 mothers (5 familial and 23 sporadic) who have an affected son with known deletion in the dystrophin gene. Only four out of 23 mothers of sporadic cases turned out to be heterozygous for the deleted exons. The lower number of carrier mothers in sporadic cases suggests a higher frequency of new mutations in North Indian DMD@BMD patients.

Dystrophin↗