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Biomedical subjects

R Dasgupta

Publications and source records attributed to R Dasgupta.

At least 19 recordsLinked to original sources
PubMed2002

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti.

Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP-N-acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5' regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

5' Untranslated Regions↗
PubMed2001

Aedes aegypti glutamine synthetase: expression and gene structure.

The peritrophic matrix (PM) is the first natural barrier a mosquito-borne parasite faces when ingested with a blood meal; consequently, understanding the biology of PM formation could provide novel transmission control strategies. Because the PM is composed of chitin (a molecule of repeating units of N-acetyl glucosamine), glycoproteins and glucose, characterizing the regulation of enzymes involved in chitin production should provide information concerning factors that influence PM formation. We previously have shown that glutamine synthetase (GS) provides the glutamine needed in the initial steps of chitin biosynthesis in the yellow fever mosquito, Aedes aegypti. In the present study we show that GS is encoded by a single 4.5 kb gene, designated mGS, containing three exons and two introns. Multiple transcripts are generated from mGS presumably by differential splicing of the introns. Sequences of two cDNAs encoding GS are identical at the protein level, but differ in their 5'-untranslated regions. GS message is constitutively expressed in all developmental stages and in most tissues, with an increase in GS transcription observed in midgut and fat body tissues of female mosquitoes following a blood meal. Transcripts are localized to the apical side of the mosquito midgut epithelium and data suggest that mGS transcription is regulated by an Oct-1 transcription factor.

Aedes↗
PubMed2001

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes.

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434-438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Biological Transport↗
PubMed2001

Diffusion with rearranging traps.

A model for diffusion on a cubic lattice with a random distribution of traps is developed. The traps are redistributed at certain time intervals. Such models are useful for describing systems showing dynamic disorder, such as ion-conducting polymers. In the present model the traps are infinite, unlike an earlier version with finite traps. For the infinite trap version a simple analytical calculation is possible and the results agree qualitatively with simulations.

Journal Article↗
PubMed2000

Early diagnosis of ectopic pregnancy: can we do it accurately using a biochemical profile?

We wanted to evaluate the utility of seven biochemical markers in the early diagnosis of ectopic pregnancy. Women with pain and bleeding suspicious for ectopic pregnancy were evaluated prospectively. Each woman had a transvaginal sonogram and serum determination of beta-human chorionic gonadotropin (beta-hCG), progesterone, estradiol (E2), creatine phosphokinase (CPK), CA-125, 17-hydroxyprogesterone, and androstenedione. The women were grouped in two ways, and there was considerable overlap between the two groups. One grouping included those with <45 days of amenorrhea, and the other included those whose beta-hCG was <2500 mIU/ml. Each marker was analyzed by univariate and multivariate logistic regression to see which could best distinguish ectopic pregnancies from nonectopic pregnancies. In the group of women with <45 days of amenorrhea, ectopic pregnancies were distinguished by a low progesterone and a high CPK. None of the other biochemical markers were significantly associated with ectopic pregnancy in the multivariate logistic regression analysis. A receiver operating characteristic (ROC) curve was constructed for the CPK/progesterone ratio. As an example of different cut points, a CPK/progesterone ratio >15 was 87% sensitive and 83% specific in the diagnosis of ectopic pregnancy. In the group of women with beta-hCG <2500 mIU/ml, the only biochemical marker found to be significantly associated with ectopic pregnancy was CPK. An ROC curve was constructed for CPK in this group. As an example of different cut points, a CPK >70 had a 78% sensitivity and 81% specificity in the diagnosis of ectopic pregnancy. We conclude that the CPK/progesterone ratio is helpful in those women with <45 days of amenorrhea, and serum CPK levels are helpful in women whose beta-hCG is <2500 mIU/ml. Although serum levels of CPK and the ratio of CPK/progesterone are the most helpful in making the diagnosis of early ectopic pregnancy, they are not good enough to be used alone in clinical practice.

17-alpha-Hydroxyprogesterone↗
PubMed1998

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Base Pair Mismatch↗
PubMed1998

Effect of nicotine use and withdrawal on brain preproenkephalin A mRNA.

Although the effect of nicotine on brain neurotransmitters and behavior has been studied, the mechanism(s) by which nicotine contributes to tobacco use remains unclear. One transmitter that may relate to long-term nicotine use and its withdrawal is enkephalin, a five-amino acid opioid peptide derived from the proenkephalin A family. In the present study we determined the effect of acute and chronic nicotine treatment and its withdrawal on preproenkephalin A mRNA levels (PPE mRNA) in specific rat brain regions using Northern blot analysis. Acute treatment with nicotine produced a significant increase in PPE mRNA in striatum and hippocampus. Chronic treatment with nicotine caused a significant decrease in PPE mRNA in these brain regions. In both striatum and hippocampus there was a rebound increase in PPE mRNA 24 h after nicotine cessation which approached the saline level 7 days later. Nicotine withdrawal 24 h following nicotine cessation, caused a significant increase in PPE mRNA in both brain regions. These effects of nicotine were blocked by pretreating rats with the nicotinic antagonist, mecamylamine. These data strongly suggest that brain opioid system(s) are involved in mediating nicotinic responses and its withdrawal and may have clinical implications in treating nicotine addiction.

Animals↗
PubMed1997

The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila.

BACKGROUND: Drosophila axis formation requires a series of inductive interactions between the oocyte and the somatic follicle cells. Early in oogenesis, Gurken protein, a member of the transforming growth factor alpha family, is produced by the oocyte to induce the adiacent follicle cells to adopt a posterior cell fate. These cells subsequently send an unidentified signal back to the oocyte to induce the formation of a polarised microtubule array that defines the anterior-posterior axis. The polarised microtubules also direct the movement of the nucleus and gurken mRNA from the posterior to the anterior of the oocyte, where Gurken signals a second time to induce the dorsal follicle cells, thereby polarising the dorsal-ventral axis. RESULTS: In addition to its previously described role in the localisation of oskar mRNA, the mago nashi gene is required in the germ line for the transduction of the polarising signal from the posterior follicle cells. Using a new in vivo marker for microtubules, we show that mago nashi mutant oocytes develop a symmetric microtubule cytoskeleton that leads to the transient localisation of bicoid mRNA to both poles. Furthermore, the oocyte nucleus often fails to migrate to the anterior, causing the second Gurken signal to be sent in the same direction as the first. This results in a novel phenotype in which the anterior of the egg is ventralised and the posterior dorsalised, demonstrating that the migration of the oocyte nucleus determines the relative orientation of the two principal axes of Drosophila. The mago nashi gene is highly conserved from plants to animals, and encodes a protein that is predominantly localised to nuclei. CONCLUSIONS: The mago nashi gene plays two essential roles in Drosophila axis formation: it is required downstream of the signal from the posterior follicle cells for the polarisation of the oocyte microtubule cytoskeleton, and has a second, independent role in the localisation of oskar mRNA to the posterior of the oocyte.

Amino Acid Sequence↗
PubMed1997

Outcome and analysis of the surgical management of esthesioneuroblastoma.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of combined radical surgical resection and radiotherapy for the treatment of esthesioneuroblastoma, a rare malignant neoplasm accounting for 1 to 5% of malignant neoplasms of the nasal cavity. METHOD: A 10-year review of 12 patients treated at our institution suggests that primary treatment consisting of a combined otolaryngologic/neurosurgical craniofacial approach results in good local control with relatively low morbidity and complication. Patients tend to present with advanced stage disease because of the nonspecific presenting signs and symptoms. Eighty-three percent of the patients were treated with combined surgery and radiation. RESULTS: Seventy-five percent of the patients are free of disease with an average follow-up time of 54 months. Based on our experience in anterior skull base surgery (zone I), we strongly advocate the pericranial flap for dural reconstruction. Postoperative complications occurred in 25% of the cases with no postoperative deaths. CONCLUSION: Failure analysis suggests that radical surgical resection combined with radiotherapy provides the best chance of disease control.

Adolescent↗
PubMed1994

Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic acid residue.

Maturation of noninfectious nodavirus provirions occurs by autoproteolytic cleavage of most of the 180 copies of the alpha-protein that make up the icosahedral capsid. This maturation, which is much slower than viral assembly, produces an infectious particle that is more stable than the provirion and makes viral uncoating thermodynamically distinct from assembly, allowing assembly and (a time-delayed) uncoating to occur under similar conditions. The results of structural, computational, and molecular genetic studies suggest that maturation depends both on intrasubunit strain, produced during assembly, and on a critical aspartic acid residue. This residue lies in a hydrophobic pocket that is stabilized by intersubunit contacts. It is close to the scissile bond and exhibits an environmentally elevated pKa. The apparent involvement of a single acidic residue in the hydrolytic cleavage of a peptide bond contrasts with the involvement of 2 such residues in acid proteases.

Aspartic Acid↗
PubMed1994

Flock house virus: a simple model for studying persistent infection in cultured Drosophila cells.

Flock house virus (FHV), isolated from twenty Drosophila melanogaster cell lines, persistently infected with the virus, were examined during successive serial passages by plaque assay and sequence analysis. No phenotypic or genotypic changes in the virus were observed during the establishment of persistent infection, suggesting that it was a cellular modification that led to the first step in establishing the persistent state. Once this state was initiated, the virus was relieved of the need for a functional coat protein to propagate itself and mutations began to accumulate selectively in RNA2, the gene for the coat protein. These changes were manifested by a gradual drift to a smaller plaque population. The replicase activity, coded by RNA1, remained unaltered.

Animals↗
PubMed1993

Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two messenger-sense RNA molecules, both of which are encapsidated in the same particle. RNA1 (3.1 kb) encodes proteins required for viral RNA replication; RNA2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein. When Spodoptera frugiperda cells were infected with a recombinant baculovirus containing a cDNA copy of RNA2, coat protein alpha assembled into viruslike precursor particles (provirions) that matured normally by autocatalytic cleavage of protein alpha into polypeptide chains beta (38 kDa) and gamma (5 kDa). The particles were morphologically indistinguishable from authentic FHV and contained RNA derived from the coat protein message. These results showed that RNA1 was required neither for virion assembly nor for maturation of provirions. Expression of mutants in which Asn-363 at the beta-gamma cleavage site of protein alpha was replaced by either aspartate, threonine, or alanine resulted in assembly of particles that were cleavage defective. For two of the mutants, unusual structural features were observed after preparation for electron microscopy. Particles containing Asp at position 363 were labile and showed a strong tendency to break into half-shells. Particles in which Asn-363 was replaced by Ala displayed a distinct hole in an otherwise complete shell. The third mutant, containing Thr at position 363, was indistinguishable in morphology from authentic FHV.

Animals↗
PubMed1992

Evidence that the packaging signal for nodaviral RNA2 is a bulged stem-loop.

Flock house virus is an insect virus belonging to the family Nodaviridae; members of this family are characterized by a small bipartite positive-stranded RNA genome. The larger genomic segment, RNA1, encodes viral replication proteins, whereas the smaller one, RNA2, encodes coat protein. Both RNAs are packaged in a single particle. A defective-interfering RNA (DI-634), isolated from a line of Drosophila cells persistently infected with Flock house virus, was used to show that a 32-base region of RNA2 (bases 186-217) is required for packaging into virions. RNA folding analysis predicted that this region forms a stem-loop structure with a 5-base loop and a 13-base-pair bulged stem.

Base Sequence↗
PubMed1990

Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography.

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.

Amino Acid Sequence↗