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Biomedical subjects

A Mukherjee

Publications and source records attributed to A Mukherjee.

At least 19 recordsLinked to original sources
PubMed2000

Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene.

During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor. We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.

Animals↗
PubMed2000

Synthesis of the tetrasaccharide related to the repeating unit of the antigen from Shigella dysenteriae type 5.

Starting from L-rhamnose, D-mannose and 2-amino-2-deoxy-D-glucose hydrochloride, two disaccharide blocks, namely, ethyl 2,4-di-O-benzyl-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha-L-rhamnopyranos yl-(1-->3)-2-O-acetyl-4,6-di-O-benzyl-1-thio-alpha-D-mannopyranoside and 2-(trimethylsilyl)ethyl 2-O-acetyl-3,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-benzy l-2-deoxy-2-phthalimido-beta-D-glucopyranoside, were synthesised and then allowed to react in the presence of N-iodosuccinimide and trifluoromethane sulfonic acid to give a tetrasaccharide derivative. This compound was converted into 2-(trimethylsilyl)ethyl 2,4-di-O-benzyl-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha-L-rhamno- pyranosyl-(1-->3)-2-O-acetyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-- >4)-2-O-acetyl-3,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-->3)-2-acetamid o-4,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside, which on hydrogenolysis, afforded the methyl ester 2-(trimethylsilyl)ethyl glycoside of the tetrasaccharide related to the repeating unit of the O-antigen from Shigella dysenteriae type 5.

Antigens, Bacterial↗
PubMed2000

Chiral nonsteroidal affinity ligands for the androgen receptor. 1. Bicalutamide analogues bearing electrophilic groups in the B aromatic ring.

A series of chiral analogues of bicalutamide bearing electrophilic groups (isothiocyanate, N-chloroacetyl, and N-bromoacetyl) on aromatic ring B of the parent molecule were synthesized. These compounds were designed as affinity ligands for the androgen receptor (AR). We prepared the (R)- and (S)-optical isomers of these compounds as pure enantiomers. The AR binding affinities of these compounds were measured in a competitive binding assay with the radiolabeled high-affinity AR ligand, [(3)H]mibolerone. In accordance with our previous results for the enantiomers of bicalutamide, we found that all (R)-isomers demonstrated much higher binding affinity to the AR as compared to their corresponding (S)-isomers. The para-substituted affinity ligands in ring B bound the AR with higher affinities than the corresponding meta-substituted analogues. Oxidation of thioester affinity ligands to their sulfonyl analogues for the para-substituted compounds decreased AR binding affinities and similar modification increased binding affinities for corresponding meta-analogues. The least potent para-substituted sulfonyl compounds had higher AR binding affinities than the most potent meta-substituted sulfonyl compounds. Overall, the para-substituted unoxidized molecules demonstrated the highest AR binding affinity. Subsequent research using AR exchange assays and Scatchard analyses showed that the isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10 reported herein are the first specific chemoaffinity ligands for the AR.

Androgen Antagonists↗
PubMed2000

The Drosophila sox gene, fish-hook, is required for postembryonic development.

In Drosophila, the fish-hook (fish) gene encodes a Sox protein essential for embryonic segmentation and nervous system organization. In this study we examined potential functional roles of fish in postembryonic developmental processes, including those involved in adult appendage development. We show here that Fish protein is expressed in discrete patterns in the larval eye-antennal and leg imaginal discs, the central nervous system, the hindgut, and salivary glands. Genetic mosaic studies indicated that fish function is required for the growth or survival of imaginal cells, and the expression of engrailed and wingless. Ectopic expression of Fish protein resulted in severe disruption of adult structures; legs and antennae were truncated and eye formation was suppressed. These morphological defects were correlated with altered expression patterns of the wingless, decapentaplegic, and bric-a-brac genes. Finally, analysis of truncated versions of Fish protein indicated that the HMG domain was sufficient for Fish nuclear localization and that removal of the transcriptional activation domain did not eliminate Fish function. While Sox proteins have been shown to be important for eye and limb formation in vertebrates, these data provide the first evidence for Sox protein functions in appendage development in invertebrates.

Animals↗
PubMed2000

In vivo cytogenetic studies on blends of aspartame and acesulfame-K.

Aspartame and acesulfame-K, non-nutritive sweeteners, are permitted individually in diets and beverages. These sweeteners of different classes, used in combination, have been found to possess a synergistic sweetening effect. Whether they also have a synergistic genotoxic effect is unknown. Swiss Albino male mice were exposed to blends of aspartame (3.5, 35, 350mg/kg body weight) and acesulfame-K (1.5, 15 and 150mg/kg body weight) by gavage. Bone marrow cells isolated from femora were analysed for chromosome aberrations. Statistical analysis of the results show that aspartame in combination with acesulfame-K is not significantly genotoxic.

Animals↗
PubMed2000

Sporotrichoid presentations in leprosy.

Two adult patients of leprosy, one woman and one man, presented with a clinical picture simulating sporotrichosis. The skin and regional nerve trunk was affected in one, and in the other the disease was confined to the nerve. Both had features of an upgrading reaction following anti-leprosy therapy; this was seen as erosion and scarring of the plaque, and acute onset of abscesses along the easily palpable and thickened nerve that ruptured through the skin. The diagnosis was supported by histopathology. In the light of other infections that give rise to a sporotrichoid pattern of infection it is concluded that leprosy should also be included in this category so that early diagnosis and use of corticosteroids can be implemented quickly to prevent nerve destruction.

Acute Disease↗
PubMed2000

Nodal metastases with unknown primary--an unusual presentation of carcinoid tumor.

Carcinoid tumors arise from neuroendocrine cells and produce their symptoms mainly through secreted amines, and by a local desmoplastic response. The critical issue in the management of advanced carcinoid tumors is preservation of quality of life and symptomatic relief. This is because of the characteristic indolent growth which makes survival a secondary consideration, and the relative rarity of the condition. Although systemic therapy has been shown to improve symptoms, true survival benefit has not been established. We present a case of carcinoid tumor that has metastasized to mesenteric nodes, and probably other locations, with an occult primary site. The patient remains completely asymptomatic and continues to enjoy a normal lifestyle. No references were found in the literature concerning the management of asymptomatic, but metastatic carcinoids. This case thus constitutes a unique situation in which further treatment may not be beneficial and may even be harmful. Nevertheless, additional treatment is an important consideration in view of the known poor survival of those with metastatic disease versus localized, resectable tumor. However, the concept of medical therapy to improve survival in an asymptomatic individual, though theoretically attractive, has little precedence. In this article we have tried to address the therapeutic dilemma posed by this unusual clinical scenario.

Aged↗
PubMed1999

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization.

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

ATP-Binding Cassette Transporters↗
PubMed1999

Affinity labeling of the androgen receptor with nonsteroidal chemoaffinity ligands.

We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.

Affinity Labels↗
PubMed1999

Hydrogeological investigation of ground water arsenic contamination in south Calcutta.

Typical clinical symptoms of acute arsenic poisoning have been detected in 1000 residents near a factory in P.N. Mitra Lane, Behala, South Calcutta, located in a thickly populated area manufacturing copper acetoarsenite (Paris-Green) an arsenical pesticide for the past 25 years. Soil around the effluent dumping point of the factory was exceptionally contaminated, with arsenic, copper and chromium concentrations of 20,100-35,500 mg kg-1, 33,900-51,100 mg kg-1 and 5300-5510 mg kg-1. Arsenic and copper concentrations in bore-hole soils collected up to a depth of 24.4 m at the effluent dumping point, decreased with depth. Arsenous acid, arsenic acid, methylarsonic acid (MA) and dimethylarsinic acid (DMA) were detected in bore-hole soils up to a depth of 1.37 m, after which only inorganic arsenical compounds were present. A positive correlation was established between arsenic and copper authenticated the Paris-Green waste disposal site as the source of contamination. Mechanism of ground water contamination from this disposal site had been probed by a systematic hydrogeological survey and the arsenic content of the tube-well waters in the surrounding areas. Hydraulic conductivity was maximum in the central part. The site for disposal of the effluent was a ditch located in the zone of discharge. Sparingly soluble Paris-Green cumulatively deposited in the waste disposal site is decomposed by micro-organisms to water-soluble forms and finally percolated to underground aquifers along with rain water through the discharge zone. The contaminant is currently moving towards WNW with ground water flow and the residents in the direction of encroaching contamination are insecure due to penetration of the contaminant.

Arsenic↗
PubMed1999

Pharmacokinetics of aminolevulinic acid after intravesical administration to dogs.

PURPOSE: To examine the stability and systemic absorption of aminolevulinic acid (ALA) in dogs during intravesical administration. METHODS: Nine dogs received an intravesical dose of ALA either with no prior treatment, after receiving ammonium chloride for urinary acidification, or after receiving sodium bicarbonate for urinary alkalinization. Urine and blood samples collected during and after administration were monitored for ALA using an HPLC assay developed in our laboratories. Concentrations of pyrazine 2,5-dipropionic acid, the major ALA degradation product, and radiolabeled inulin, a nonabsorbable marker for urine volume, were also determined. RESULTS: Less than 0.6% of intravesical ALA doses was absorbed into plasma. Urine concentrations decreased to 37% of the initial concentration during the 2 hour instillation. Decreases in urinary ALA and radiolabeled inulin concentrations were significantly correlated, indicating that urine dilution accounted for over 80% of observed decreases in urinary ALA. ALA conversion to pyrazine 2,5-dipropionic acid was negligible. CONCLUSIONS: These studies demonstrate that ALA is stable and poorly absorbed into the systemic circulation during intravesical instillation. Future studies utilizing intravesical ALA for photodiagnosis of bladder cancer should include measures to restrict fluid intake as a means to limit dilution and maximize ALA concentrations during instillation.

Administration, Intravesical↗
PubMed1999

Recruitment of ZipA to the division site by interaction with FtsZ.

ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle. As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring. In this study, we have investigated the requirements for ZipA localization to the division site. ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ. Consistent with this, apparently normal Z rings are formed in the absence of ZipA. The interaction between FtsZ and ZipA occurs through their carboxy-terminal domains. Although a MalE-ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments. These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.

ATP-Binding Cassette Transporters↗
PubMed1999

rsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin(Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA).

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin(Ecc) (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219-234, 1998). We have cloned and characterized a novel regulatory gene, rsmC, that activates RsmA production and represses extracellular enzyme and harpin(Ecc) production, rsmB transcription, and virulence in E. carotovora subsp. carotovora. In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA(+) allele, the basal levels of Pel, Peh, Cel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, and hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC(-) mutant than in the RsmC(+) parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC(-) mutant than in the RsmC(+) parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmA(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutant is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora. These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.

Amino Acid Sequence↗
PubMed1999

Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations.

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90 degrees angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg2+ was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca2+ mediated dynamic assembly of FtsZ (X. -C. Yu and W. Margolin, EMBO J. 16:5455-5463, 1997). However, we found that Ca2+ was not required for FtsZ assembly and that this concentration of Ca2+ reduced the dynamic behavior of FtsZ assembly.

Bacterial Proteins↗
PubMed1999

kdgREcc negatively regulates genes for pectinases, cellulase, protease, HarpinEcc, and a global RNA regulator in Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.

Amino Acid Sequence↗
PubMed1999

Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult.

Targeted disruption of the mouse estrogen receptor-alpha gene (estrogen receptor-alpha knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ER alpha-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA expression was also higher in the ERKO animals. Cellular localization studies by in situ hybridization analysis of ERKO ovaries showed a high level of LHR mRNA expression in the granulosa and thecal layers of virtually all the antral follicles. Ribonuclease protection analyses showed that ovarian progesterone receptor and androgen receptor mRNA expression were similar in the two groups. These results indicated that ER alpha action was not a prerequisite for LHR mRNA expression by thecal or granulosa cells or for ovarian expression of progesterone receptor mRNA. Ovarian estrogen receptor beta (ER beta) was detected immunohistochemically, was sharply compartmentalized to the granulosa cells, and was expressed approximately equally in the ERKO animals and the WT controls. In contrast, ER alpha staining was present in the thecal cells but not the granulosa cells of the WT animals. The summary findings indicate that in the adult the major cause of the ERKO phenotype is high circulating LH interacting with functional LHR of the theca and granulosa cells. These features result in a failure of the normal maturational events leading to successful ovulation and luteinization and presumably involve both hypothalamic-pituitary and intraovarian mechanisms dependent upon ER alpha action. The presence of ER beta in the granulosa cells did not rescue the phenotype of the ovary.

Animals↗