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Biomedical subjects

B Sarkar

Publications and source records attributed to B Sarkar.

At least 19 recordsLinked to original sources
PubMed2004

Effects of cypermethrin and carbofuran on certain hematological parameters and prediction of their recovery in a freshwater teleost, Labeo rohita (Hamilton).

Sublethal effects of cypermethrin and carbofuran on hematological parameters and their complete recovery were studied in Labeo rohita as a function of exposure time. Exposure of Labeo rohita to sublethal levels of cypermethrin and carbofuran resulted in significantly (P<0.05) lower values for erythrocyte count (RBC), hemoglobin content (Hb), and hematocrit compared with the control group. In contrast, there was a significant increase (P<0.05) in leukocyte count (TLC) in the pesticide-treated group. Mean cell volume (MCV) and mean cell hemoglobin (MCH) increased in response to both pesticides during the study. The rate of recovery or extrapolation time between Days 0 and 28 of the recovery period revealed that RBC completely recovered from cypermethrin toxicity on Days 80, 86.6, and 90 and TLC recovered on Days 60, 62.7, and 63.3 in fish exposed to 0.16, 0.40, and 0.80 microL/L cypermethrin, respectively. In the case of carbofuran toxicity, the complete recovery for RBC was on Days 100, 101.5, and 102, and for TLC, on Days 77, 77.6, and 79.3 in fish exposed to 0.06, 0.15, and 0.30 mg/L carbofuran, respectively. TLC recovered faster than RBC. RBC and TLC of fish exposed to carbofuran took more time to complete recovery than RBC and TLC in fish exposed to cypermethrin.

Animals↗
PubMed2002

Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup.

Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.

Animals↗
PubMed2001

Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy.

The use of a short, three-residue Cu(2+)-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu(2+) can be converted into distance restraints and used for structure refinement by using a new routine, PMAG, that has been written for the structure calculation program CNS. The utility of the approach is demonstrated with an application to ATCUN-tagged ubiquitin. Excellent agreement between measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.

Amino Acid Motifs↗
PubMed2001

Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli.

ATP7B, the Wilson disease-associated Cu(I)-transporter, and ZntA from Escherichia coli are soft metal P1-type ATPases with mutually exclusive metal ion substrates. P1-type ATPases have a distinctive amino-terminal domain containing the conserved metal-binding motif GXXCXXC. ZntA has one copy of this motif while ATP7B has six copies. The effect of interchanging the amino-terminal domains of ATP7B and ZntA was investigated. Chimeric proteins were constructed in which either the entire amino-terminal domain of ATP7B or only its sixth metal-binding motif replaced the amino-terminal domain of ZntA. Both chimeras conferred resistance to lead, zinc, and cadmium salts but not to copper salts. The purified chimeras displayed activity with lead, cadmium, zinc, and mercury, which are substrates of ZntA. There was no activity with copper or silver, which are substrates of ATP7B. The chimeras were 2-3-fold less active than ZntA. Thus, the amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. Also, these results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity.

Adenosine Triphosphatases↗
PubMed2000

Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase.

The Wilson disease copper-transporting ATPase plays a critical role in the intracellular trafficking of copper. Mutations in this protein lead to the accumulation of a toxic level of copper in the liver, kidney, and brain followed by extensive tissue damage and death. The ATPase has a novel amino-terminal domain ( approximately 70 kDa) which contains six repeats of the copper binding motif GMTCXXC. We have expressed and characterized this domain with respect to the copper binding sites and the conformational consequences of copper binding. A detailed analysis of this domain by X-ray absorption spectroscopy (XAS) has revealed that each binding site ligates copper in the +1 oxidation state using two cysteine side chains with distorted linear geometry. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the function and regulation of the ATPase in vivo. In addition to providing important insights on copper binding to the protein, these results suggest a possible mechanism of copper trafficking by the Wilson disease ATPase.

Adenosine Triphosphatases↗
PubMed2000

Window rectostomy: an alternative method of fecal diversion for high anorectal malformations in males without a urinary fistula.

This report analyzes our initial experience with window rectostomy (WR) as a new method of fecal diversion for high anorectal malformations (ARM) in 27 males without a urinary fistula between May 1994 and May 1998; total correction was achieved in two stages. In the first stage, during the neonatal period, the dilated rectum was exteriorized as a WR through the left lower abdomen. In the second, after 3-5 months an abdominoperineal pull-through (APPT) procedure was performed in which the window rectostomy was mobilized and taken down to form a new anus. The results were compared with cases of high ARM with urinary fistula that were managed in three stages, i.e., proximal sigmoid colostomy, APPT, and colostomy closure. All 27 cases showed satisfactory results without any mortality or major pelvic infection. The main advantages of WR are that it provides more functioning bowel length after diversion and avoids a colostomy-closure operation and repeated hospitalizations, thus reducing the total cost of treatment.

Anal Canal↗
PubMed2000

Copper transport and its defect in Wilson disease: characterization of the copper-binding domain of Wilson disease ATPase.

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. An attempt is made here to present the current understanding of the normal transport of copper in relation to the absorption, intracellular transport and toxicity. Wilson disease is a genetic disorder of copper transport resulting in the accumulation of copper in organs such as liver and brain which leads to progressive hepatic and neurological damage. The gene responsible for Wilson disease (ATP7B) is predicted to encode a putative copper-transporting P-type ATPase. An important feature of this ATPase is the presence of a large N-terminal domain that contains six repeats of a copper-binding motif which is thought to be responsible for binding this metal prior to its transport across the membrane. We have cloned, expressed and purified the N-terminal domain (approximately 70 kD) of Wilson disease ATPase. Metal-binding properties of the domain showed the protein to bind several metals besides copper; however, copper has a higher affinity for the domain. The copper is bound to the domain in Cu(I) form with a copper: protein ratio of 6.5:1. X-ray absorption studies strongly suggest Cu(I) atoms are ligated to cysteine residues. Circular dichroism spectral analyses suggest both secondary and tertiary structural changes upon copper binding to the domain. Copper-binding studies suggest some degree of cooperativity in binding of copper. These studies as well as detailed structural information of the copper-binding domain will be crucial in determining the specific role played by the copper-transporting ATPase in the homeostatic control of copper in the body and how the transport of copper is interrupted by mutations in the ATPase gene.

Adenosine Triphosphatases↗
PubMed2000

Posterior sagittal approach for repair of rectourethral fistula occurring after perineal surgery for imperforated anus at birth.

PURPOSE: Posterior sagittal approach was used for the repair of 11 cases of rectourethral fistula between 1992 and 1998. METHODS: All these patients had rectourethral fistula as a result of perineal procedure performed for imperforate anus at the time of birth. In addition to the fistula repair, an associated anorectal stenosis (3 cases) and urethral stricture 11 case) also were corrected with this approach. All the patients underwent surgery under cover of a protective colostomy. RESULTS: Successful repair was achieved in all patients, and the follow-up period ranged from 10 months to 7 years. As far as bladder and bowel control, urinary control was normal in all 11 patients, whereas bowel control was normal in 10 cases. One patient who had severe scarring of anal sphincters caused by infection in the previous surgery still suffers occasional perianal soiling after 18 months of follow-up. CONCLUSIONS: Posterior sagittal approach not only gives adequate exposure but also suits the basic principles of fistula repair, namely, completely separating the rectum from urethra and leaving normal rectal wall behind the urethral sutures thus eliminating the possibility of recurrence. In addition to fistula repair, one can also correct associated problems like anorectal stenosis or urethral stricture, and a mislocated rectum can be relocated within the sphincter complex.

Adult↗
PubMed1999

Addition of positively charged tripeptide to N-terminus of the Fos basic region leucine zipper domain: implications on DNA bending, affinity, and specificity.

GKH-Fos(139-211)/Jun(248-334) (GKH: glycine-lysine-histidine) is a modified Fos/Jun heterodimer designed to contain a metal binding motif in the form of a GKH tripeptide at the amino terminus of Fos bZIP domain dimerized with the Jun basic region leucine zipper (bZIP) domain. We examined the effect of the addition of positively charged GKH motif to the N-terminus of Fos(139-211) on the DNA binding characteristics of the Fos(139-211)/Jun(248-334) heterodimer. Binding studies indicate that while the nonspecific DNA binding affinity of the GKH modified heterodimer increases 4-fold, it specifically binds the activating protein-1 (AP-1) site 6-fold less tightly than the control unmodified counterpart. Furthermore, helical phasing analysis indicates that GKH-Fos(139-211)/Jun(248-334) and control Fos(139-211)/Jun(248-334) both bend the DNA at the AP-1 site toward the minor groove. However, due to the presence of the positively charged GKH motif on Fos, the degree of the induced bend by GKH- Fos(139-211)/Jun(248-334) is greater than that induced by the unmodified Fos/Jun heterodimer. Our results suggest that the unfavorable energetic cost of the increased DNA bending by GKH-Fos(139-211)/Jun(248-334) results in a decrease in both specificity and affinity of binding of the heterodimer to the AP-1 site. These findings may have important implications in protein design as well in our understanding of DNA bending and factors responsible for the functional specificity of different members of the bZIP family of transcription factors.

Amino Acid Sequence↗
PubMed1998

Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition.

Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation. Many of these zinc-finger domains are involved in DNA binding. This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition. (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition. The protein-DNA interaction was examined in greater detail by using a methylation-interference assay. The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide. This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers.

Amino Acid Sequence↗
PubMed1998

Early treatment of Menkes disease with parenteral copper-histidine: long-term follow-up of four treated patients.

We report on the long-term clinical course of 4 boys with Menkes disease, treated from early infancy with parenteral copper-histidine, with follow-up over 10-20 years. Three of the 4 had male relatives with a severe clinical course compatible with classical Menkes disease. As a consequence of early treatment, our patients have normal or near-normal intellectual development, but have developed many of the more severe somatic abnormalities of the related disorder, occipital horn syndrome, including severe orthostatic hypotension in 2. In addition, 1 boy developed a previously unreported anomaly, namely, massive splenomegaly and hypersplenism as a consequence of a splenic artery aneurysm. Previously reported molecular studies in 2 of these patients had shown gene defects which would have predicted a truncated and probably nonfunctional gene product. Despite the favorable effects on the neurological symptoms, parenteral copper treatment for Menkes disease should still be regarded as experimental. The development of more effective treatments must await a more precise delineation of the role which the Menkes protein plays in intracellular copper trafficking.

Adolescent↗
PubMed1998

Bazex syndrome (acrokeratosis paraneoplastica).

Acrokeratosis paraneoplastica (Bazex syndrome) is a rare but distinctive dermatosis associated with carcinomas of the upper aerodigestive tract with possible cervical lymph node metastases. The cutaneous lesions occurring in the syndrome are non-metastatic skin involvement that parallels (as cutaneous marker) the evolution of the malignancy. Since only a few case reports have been published in the otolaryngologic literature, we present our experience and review some of the findings previously reported.

Acrodermatitis↗
PubMed1998

Management of congenital and acquired H-type anorectal fistulae in girls by anterior sagittal anorectovaginoplasty.

METHODS: Thirteen girls with congenital or acquired H-type anorectal fistulae underwent surgery between 1991 and 1996. In all cases, besides a normally placed anal canal, there was a fistulous communication between the anorectum and the genital tract. On the basis of the level of fistulous communication, these cases were divided into three groups: high, intermediate, and low (perineal canal). All patients underwent anterior sagittal anorectovaginoplasty. Surgical technique included division of all intervening tissue in midline between the perineal skin and the fistula. The whole fistulous tract was excised, and the remaining surrounding tissue was repaired in different layers. Of 13 patients, 12 were operated on without a protective colostomy. RESULTS: There was no recurrence in any case, and all patients had good cosmetic results with a normal sphincter control. Although various techniques have been suggested for the surgical correction of H-type anorectal fistulae, most of them are applicable only to the low-lying fistula (perineal canal). CONCLUSIONS: To date, there is no satisfactory method available for correction of high fistula. The methods suggested for high fistula (abdominoperineal pull-through and endorectal pull-through) appear to be too extensive for this condition. Our technique of anterior sagittal anorectovaginoplasty can be used not only for low fistula but can also be used for intermediate and high types of fistulae. This technique is simple, safe, takes less time, and achieves good anatomic and functional reconstruction of the perineum.

Adolescent↗
PubMed1998

Effect of redox conditions on the DNA-binding efficiency of the retinoic acid receptor zinc-finger.

Retinoic acid and its derivatives are involved in many important biological processes. In the present study, we have shown that the DNA binding domain of the retinoic acid receptor, which contains two zinc fingers with the Zn(II) tetrahedrally coordinated by four Cys, is susceptible to intracellularly relevant oxidizing agents. In the presence of hydrogen peroxide or hypochlorite, the zinc-finger DNA binding activity was abolished in a concentration dependent manner. The loss of DNA binding activity was correlated with the release of Zn(II) from the zinc-finger motif as a consequence of Zn(II)-thiolate bond oxidation. A combination of glutathione and Zn(II) was able to restore the activity, suggesting that oxidation of the zinc-finger by hydrogen peroxide or hypochlorite resulted in the formation of disulfide bonds between the Cys present in the Zn(II)-binding motif. Our results indicate that in situations of oxidative-stress zinc-finger containing transcription factors may be particularly susceptible to oxidation, resulting in the disruption of control and regulation of gene expression.

Animals↗
PubMed1997

Expression, purification, and metal binding properties of the N-terminal domain from the wilson disease putative copper-transporting ATPase (ATP7B).

The putative copper binding domain from the copper-transporting ATPase implicated in Wilson disease (ATP7B) has been expressed and purified as a fusion to glutathione S-transferase. Immobilized metal ion affinity chromatography revealed that the fusion protein is able to bind to columns charged with different transition metals with varying affinities as follows: Cu(II)>>Zn(II)>Ni(II)>Co(II). The fusion protein did not bind to columns charged with Fe(II) or Fe(III). 65Zinc(II) blotting analysis showed that the domain is able to bind Zn(II) over a range of pH values from 6.5 to 9.0. Competition 65Zn(II) blotting showed that Cd(II), Hg(II), Au(III), and Fe(III) can successfully compete with Zn(II), at comparable concentrations, for binding to the domain. In contrast, the domain had little or no affinity for Ca(II), Mg(II), Mn(II), and Ni(II) relative to copper. Neutron activation analysis of the copper bound to the domain showed a copper:protein ratio of 6.5-7.3:1. Both Cu(II) and Cu(I) were found to have a higher affinity for the domain relative to Zn(II). In addition, a sharp, reproducible transition was only observed in competition experiments with copper, which may suggest that copper binding has some degree of cooperativity.

Adenosine Triphosphatases↗