Search PubMed⌕ Search

Biomedical subjects

S Sinha

Publications and source records attributed to S Sinha.

At least 289 records · Page 16Linked to original sources
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↗
PubMed1996

Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

Amino Acid Sequence↗
PubMed1996

Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule.

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Amino Acid Sequence↗
PubMed1996

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

Animals↗
PubMed1996

Spontaneous rupture of spleen in viral hepatitis A.

A twenty-five year old man presented with history of fever, malaise and jaundice and developed a spontaneous rupture of the spleen during hospital admission. Investigations revealed deranged liver function tests and positive serology for hepatitis A.

Adult↗
PubMed1996

Alterations in brain tumor DNA detected by a fingerprinting probe.

We used a novel DNA fingerprinting probe O-chi-1 (ref. 1) to detect differences in the hybridization pattern of brain tumor DNA and paired normal tissue of a given individual. Representatives of meningiomas (two), glioblastoma multeforme (three) and astrocytoma (one) were studied. Alterations, which included amplification as well as the loss of a normal band in tumor DNA, were observed in four of the six tumours. While the increased intensity of a band can be taken to imply increased copy number, the disappearance of bands could either be due to loss of DNA sequence or rearrangement resulting in different sized bands.

Astrocytoma↗
PubMed1995

Infrequent alteration of the c-myc gene in human glial tumours associated with increased numbers of c-myc positive cells.

Twenty five human glial tumours of different grades of malignancy were examined by Southern blotting and polymerase chain reaction (PCR) for alterations (rearrangements, amplification and deletions) in the c-myc gene. Number of c-myc positive cells per thousand cells were also counted in all the tumours after immunohistochemical staining for c-myc protein was done on fixed sections of the tumours. No tumours exhibited any amplification of the gene, as found by Southern blotting. One astrocytoma and one mixed glioma showed some rearrangements in the 3' end of the gene, as detected by Southern blotting and hydridization. These two tumours had higher number of c-myc positive cells than in other tumours of the same histopathological groups. Deletion in the first promoter region, as determined by PCR, was seen in only one astrocytoma. However, the number of c-myc positive cells in that tumour did not show any deviation from that found in other astrocytomas. In light of present literature, it is speculated that the 3' rearrangements may be the cause of increased number of c-myc immunopositive cells in those tumours by disrupting the 3' end of the gene leading to increased c-myc mRNA stability. Such a mechanism may play a part in small subset of glial and possibly other tumours.

Base Sequence↗
PubMed1995

Cardiovascular and respiratory changes following exposure to a synthetic toxin of Ptychodiscus brevis.

The present study demonstrated cardiorespiratory effects of a synthetic phosphorus-containing ichthyotoxic metabolite elaborated by the marine dinoflagellate Ptychodiscus brevis in anaesthetised cats. The metabolite at a dose of 0.25-1.5 mg/kg i.v., resulted in a dose-dependent fall in blood pressure and such vasodepressor effect was associated with bradycardia. There is initial respiratory apnoea followed by increased rate and depth of respiration (hyperapnoea) following the administration of the toxin. The hypotensive response was accompanied by a decrease in aortic baroreceptor activity. The ECG showed atrioventricular conduction block, arrhythmia and depression of S-T segment and T wave which indicated coronary insufficiency. Vasodepressive property of the toxin is presumably muscarinic in nature as atropine counteracted the vasodepression.

Animals↗
PubMed1995

Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3.

The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.

Amino Acid Sequence↗
PubMed1995

Cell-type and amyloid precursor protein-type specific inhibition of A beta release by bafilomycin A1, a selective inhibitor of vacuolar ATPases.

Treatment of human 293 cells transfected with amyloid precursor protein (APP)K595N,M596L (the "Swedish" mutation) with a specific inhibitor of the vacuolar H(+)-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the A beta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of A beta from either wild-type APP (WT APP) or from APPv7171 (the "Hardy" mutation), expressed in the same cell type. In contrast, the robust production of A beta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of A beta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and A beta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in A beta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and A beta release could be one explanation for the increased production of A beta observed on expression of this mutation.

Adenosine Triphosphatases↗
PubMed1995

Species-specific 18S rRNA gene amplification for the detection of P. falciparum and P. vivax malaria parasites.

Based on the sequence diversity of the Plasmodium 18S ribosomal RNA (rRNA), we designed oligonucleotide primers for polymerase chain reaction (PCR) to yield different size fragments for P. falciparum and P. vivax. The primers for the PCR procedure were chosen such that the 5' primer was Plasmodium-conserved while the 3' primers were species-specific. Using primer cocktails and cloned plasmid DNAs containing the 18S rRNA genes or parasite genomic DNA as targets, we show that the PCR procedure yields 1.4-kb and 0.5-kb DNA fragments for P. falciparum and P. vivax, respectively. Limited field testing of this procedure demonstrated the utility of a ribosomal gene based species-specific malaria diagnosis.

Animals↗
PubMed1995

Estimation of gestational maturity of preterm infants by five fetal sonographic measurements compared with neonatal EEG and the last menstrual period.

We previously reported no differences in estimates of gestational maturity between a cumulative score based on sonographic/obstetrical measurements and an EEG interpretation for healthy preterm neonates. For the present study, a rank order among 5 fetal sonographic measurements was used to estimate gestational age: head circumference, transcerebellar diameter, biparietal diameter, femur length, and abdominal circumference were compared with an electroencephalographic estimate and an arithmetic estimate of maturity (i.e., the mother's last menstrual period). A fetal sonographic study for each of 13 premature neonates (i.e., born at < 32 weeks estimated gestational age) was followed after birth by a neonatal EEG recording. EEG and sonographic estimates of maturity were assigned without knowledge of other clinical data. The mother's last menstrual period was obtained from the medical record. Principal component analysis determined that 86% of the variance was evenly distributed over the 5 sonographic measurements. Using a regression procedure, 61% of the variance (adjusted r2 = 0.61) was explained by EEG when compared with fetal sonographic estimates. Only 19% (adjusted r2 = 0.19) of the variance was explained by the estimate based on the mother's last menstrual period. Optimal subset selection determined that the rank order for prediction of gestational maturity among the fetal sonographic measurements was as follows: (1) head circumference; (2) transcerebellar diameter; (3) biparietal diameter; (4) femur length; (5) abdominal circumference. Cranial measurements on fetal sonography (i.e., head circumference, transcerebellar diameter, biparietal diameter) compared more closely with EEG estimates than non-cranial measurements (i.e., femur length and abdominal circumference). In conclusion, neonatal EEG estimates of gestational maturity compared more closely with sonographic measurements than estimates based on the time of the last menstrual period. These findings will be useful in situations when dates of conception are unknown or inaccurate, or when intrauterine growth restriction causes discrepancies in growth rates between cranial and non-cranial fetal measurements on sonography.

Brain↗