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Biomedical subjects

K Majumder

Publications and source records attributed to K Majumder.

18 recordsLinked to original sources
PubMed2006

Menopausal symptoms in women treated for breast cancer: the prevalence and severity of symptoms and their perceived effects on quality of life.

OBJECTIVES: To determine, first, the prevalence and severity of various symptoms related to estrogen deficiency in women within a few years of receiving treatment for breast cancer, second, how women perceive the effects of these symptoms on their quality of life and, third, what measures have been taken to relieve vasomotor symptoms. METHODS: Two hundred women (aged 29-65 years) who had received treatment for breast cancer within the last 5 years were included in this cross-sectional survey. Information was collected about their breast cancer treatment, menopausal symptoms (Menopausal Rating Scale), the perceived effects of menopausal symptoms on their and their partner's quality of life and any treatments they were receiving for hot flushes. RESULTS: All but one woman reported at least one symptom related to the menopause (95.9% vasomotor; 83.3% psychological; 89.7% somatic). Current treatment with tamoxifen or previous chemotherapy did not influence the prevalence or the severity of hot flushes. Current antidepressant treatment was, however, significantly associated with a higher prevalence and severity of most menopausal symptoms, including hot flushes and sweats (p = 0.008). The severity of hot flushes and sweats was significantly correlated with self-assessed effects on overall quality of life (r(s) = 0.47); 56.4% of the respondents believed that menopausal symptoms had affected their partner's quality of life, the strongest correlations being with severity of sexual symptoms (r(s) = 0.56) and vaginal dryness (r(s) = 0.5). Only 21% of women experiencing hot flushes were receiving any treatment for hot flushes, with most women describing no knowledge or poor knowledge of treatment options. CONCLUSIONS: The majority of women receiving treatment for breast cancer report menopausal symptoms, which negatively correlate, not only with their own, but also with their partner's quality of life. Most women experiencing hot flushes are not receiving treatment due to lack of both awareness and confidence in the existing treatment options.

Adult↗
PubMed1998

Differential expression of flectin in the extracellular matrix and left-right asymmetry in mouse embryonic heart during looping stages.

A novel extracellular matrix protein flectin (250 kD M(r)) shows specific left-right asymmetric expression before and throughout the looping process during heart development in avian embryos [Tsuda et al., 1996]. Flectin is a candidate molecule to provide directionality to the looping process in the avian model. In this study on mouse embryonic heart development, flectin is shown to be developmentally regulated and to be expressed in a specific asymmetric fashion, but in a different pattern from that observed in avian hearts. The molecules involved in development tend to be the same, but timing of expression, modulation, and asymmetry are different. In the mouse embryo, flectin is expressed symmetrically when the cardiogenic plate is formed. As looping progresses, flectin expression becomes asymmetric. There is right side predominance at the outflow tract and left side predominance at the ventricular portion of the tubular heart. The left side predominance of flectin develops in an anteroposterior direction, while right side predominance of the outflow tract remains relatively unchanged. These differential expression patterns of flectin decrease once the looping process is completed. After looping, flectin becomes restricted to the epicardium and subepicardial extracellular regions. In inv/inv mice, a known mouse model for human situs inversus, in which the directionality of heart looping is inverted, flectin expression pattern is mirror image of that of normal mouse embryos during looping stages. Our study indicates that, in the mouse, flectin shows a specific asymmetric expression pattern after initiation of heart looping and that this asymmetric expression pattern is related to the directionality of looping. The remodeling of the extracellular matrix (ECM) including specific flectin expression begins with the looping process. This morphogenetic change of the ECM coincides with the differentiation of each region of the tubular heart.

Animals↗
PubMed1998

YAC rescue of downless locus mutations in mice.

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dlslk) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type locus.

Alleles↗
PubMed1998

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).

Amino Acid Sequence↗
PubMed1996

Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis.

The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site-directed mutagenesis. Native and the mutant proteins were expressed using a two-vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis.

The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants. Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis is an allosteric enzyme. Analysis of Arg24-->Asp, Pro105-->Ser and His385-->Lys mutations suggests a hidden phosphoenolpyruvate-binding site.

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N-terminal, middle and C-terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis. Steady-state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimate-3-phosphate synthase enzymes investigated so far. Both substrates, phosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multiple interaction sites. There are two sites for P-pyruvate binding, catalytic and non-catalytic. Glyphosate (N-phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P-pyruvate binding and favors dimerization of the enzyme through an interaction between P-pyruvate-binding sites. The ammonium-ion-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24-->Asp and His385-->Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium.

We report the cloning and functional expression of a novel K+ channel beta-subunit from human atrium, hKv beta 3. hKv beta 3 is highly homologous to the two beta-subunits cloned from rat brain, Kv beta 1 and Kv beta 2, but has an essentially unique stretch of 79 N-terminal residues. Upon expression in Xenopus oocytes, hKv beta 3 accelerates the inactivation of co-injected hKv1.4 currents and induces fast inactivation of non-inactivating co-injected hKv1.5 currents. By contrast, hKv beta 3 had no effect on hKv1.1, hKv1.2, or hKv2.1 currents. Thus, hKv beta 3 represents a third type of K+ channel beta-subunit which modulates the kinetics of a unique subset of channels in the Kv1 subfamily.

Amino Acid Sequence↗
PubMed1995

Background-minimized cassette mutagenesis by PCR using cassette-specific selection markers: a useful general approach for studying structure-function relationships of multisubstrate enzymes.

An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA. This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci. Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process. First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s). Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants. Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods. The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system. Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs. Very high efficiency of mutation incorporation and selection were obtained in all cases. Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants. The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Cloning and functional expression of an inwardly rectifying K+ channel from human atrium.

The cardiac inward rectifier current (IK1) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K+ channels (IRKs) from different tissues have been cloned recently, the nature and number of K+ channels contributing to the cardiac IK1 are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase-polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited < 70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in Xenopus oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac IK1 in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.

Amino Acid Sequence↗
PubMed1994

Recombinant enrichment by exploitation of restriction sites with interrupted palindromes: design, synthesis and incorporation of zero-background linkers in cloning and expression vectors.

A universal approach for improving the efficiency of cloning through a selective enrichment of recombinants has been developed. This was achieved by using novel polylinkers or multiple cloning sites (MCS) termed zero-background linkers (ZBL). These MCS have short-cutter site(s) incorporated in the wobble portion of the recognition sequence of the long-cutter(s) in such a fashion that cloning at the short-cutter site disrupts the continuity of the long-cutter-specific sequence. Consequently, digestion of the ligation mixture with the long-cutter, prior to transformation, essentially eliminates the insert negative clones from transformants. The usefulness of such background removal has been illustrated through the design and synthesis of a model ZBL molecule and then its incorporation into various popular cloning and expression vectors. We demonstrate that replacement of the residing MCS of a vector with ZBL leads to enhanced cloning efficiency, as evidenced by the marked increase in the ratio of recombinants to non-recombinants. Such vector improvement is nearly universal as zero backgrounding is an add-on feature and can be adapted to practically any vector system, without having to alter the essential features of the parent vectors.

Base Sequence↗
PubMed1993

Solution structure of GCCAAT recognition motif by 2D NMR, spectral simulation, molecular modeling, and distance geometry calculations.

Solution conformation of a self-complementary 14-mer DNA duplex (d-GGATTGGCCAATCC) containing the GCCAAT recognition motif of several transcription factors has been investigated by NMR spectroscopy. Complete resonance assignment of all the protons (except H5',H5'' protons) has been obtained following standard procedures based on two-dimensional NMR techniques. Three-bond coupling constants have been determined by spectral simulation procedures. New strategies have been described and employed for quantifying NOE intensities from the structural point of view. Approximate ranges of gamma torsion angles have been obtained from a selective NOESY experiment, by estimating the J(4'-5'), J(4'-5''), or their sum in the H1'-H4' cross peaks of the spectrum. Likewise, ranges of epsilon torsion angles have been obtained by monitoring the H3' multiplicities in the H8/H6-H3' cross peaks in selective NOESY spectra. With the help of such a total of 73 coupling constraints, 79 NOE intensity constraints, and 108 H-bond constraints, model building has been carried out to obtain a structure which satisfies the constraints. Starting from such a structure, an expanded distance constraint set has been created which has been used for the distance geometry calculations using the program TANDY. In the best structure thus derived, interesting irregularities similar to a BI-BII transition have been observed in the center. The molecule exhibits a bend. The overall base stacking is different from that in either B- or A-DNA models. The base pairs are tilted with respect to the local helix axes. The observed structural features are likely to have important implications for the recognition mechanism of the GCCAAT motif.

Algorithms↗
PubMed1992

Ligation-free gene synthesis by PCR: synthesis and mutagenesis at multiple loci of a chimeric gene encoding OmpA signal peptide and hirudin.

A unique kination and ligation-free method that allows de novo synthesis of a gene through a novel application of polymerase chain reaction (PCR) involving stepwise elongation of sequence (SES) is described. SES-PCR is simple and efficient. Optimal utilization of nucleotides, ability to use only partially purified oligodeoxyribonucleotides, and elimination of kination and ligation of intermediates make SES-PCR-mediated gene synthesis more economical in terms of time, labour and money. Site-directed mutagenesis and/or gene fusion by SES-PCR is not limited by the prior availability of the gene(s) in question. The potentials of this novel method in gene synthesis, mutagenesis at multiple loci of DNA and gene fusion have been demonstrated using a chimeric gene encoding fusion between OmpA signal peptide and hirudin, as an example. The SES-PCR product was cloned and sequencing of positive clones demonstrated the presence of genes with expected sequence and bearing only the desired mutations. A nearly 100% efficiency of mutation was easily achieved by the design of the method.

Amino Acid Sequence↗
PubMed1989

Sequence specificity in spermine-induced structural changes in CG-oligomers.

The role of spermine in inducing A-DNA conformation in deoxyoligonucleotides has been studied using CCGG and GGCC as model sequences. It has been found that while CCGG adopts an alternating B-DNA conformation in low salt solution at low temperature, addition of spermine to this medium induces a B --greater than A transition. In contrast, the A-DNA-like structure of GGCC in low salt solution at low temperature does not change under the influence of spermine. This suggests a sequence-dependent behaviour of spermine. Further these results suggest that the A-DNA conformation observed in the crystals of d(iCCGG) and d(GGCC)2 might have been due to the presence of spermine in the crystallization cocktail.

Circular Dichroism↗
PubMed1988

Sequence specificity of Z-DNA formation in oligonucleotides.

The sequence specific requirement for B----Z transition in solution was examined in d(CGTGCGCACG), d(CGTACGTACG), d(ACGTACGT) in presence of various Z-inducing factors. Conformational studies show that inspite of the alternating nature of purines and pyrimidines, the aforementioned sequences do not undergo B----Z transition under the influence of NaCl, hexamine cobalt chloride and ethanol. A comparison with the crystal structures of an assorted array of purine and pyrimidine sequences show that the sequence requirement for B----Z transition is much more stringent in solution as compared to the solid state. The disruptive influence of AT base pairs in B to Z transition is discussed.

Base Sequence↗