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Biomedical subjects

K Chowdhury

Publications and source records attributed to K Chowdhury.

At least 19 recordsLinked to original sources
PubMed2001

Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The Spnr mRNA is expressed at high levels in testis, ovary, and brain and is present in these tissues in multiple forms. We have generated a gene trap allele of the murine Spnr, named Spnr(+/GT). Spnr(GT/GT) mutants show a high rate of mortality, reduced weight, and an abnormal clutching reflex. In addition to minor anatomical abnormalities in the brain, males exhibit defects in spermatogenesis that include a thin seminiferous epithelium and disorganization of spermatogenesis. Most of the sperm from mutant males display defects in the flagellum and consequently show decreased motility and transport within the oviducts. Furthermore, sperm from mutant males achieve in vitro fertilization less frequently. Our findings suggest that SPNR plays an important role in normal spermatogenesis and sperm function. Thus, the Spnr(GT/GT) mutant male mouse provides a unique model for some human male infertility cases.

Alleles↗
PubMed2001

Pax4 regulatory elements mediate beta cell specific expression in the pancreas.

Pax4 is a member of the Pax (Pax, paired box) family of transcription factors with restricted expression and essential functions in the developing pancreas. Pax4 deficient mice do not develop pancreatic beta and delta cells and thus die after birth due to diabetes. In this study using transgenic mouse technology we report the identification and characterization of a 0.9 kb DNA fragment in the 5'-region of the gene, which by itself is able to direct Pax4 expression in the endocrine pancreas, recapitulating the beta-cell-specific in vivo expression pattern of Pax4. The 0.9 kb DNA fragment contains an area spanning 407 base pairs that is highly conserved between human and mouse showing 88% identity. This promoter region contains sequence motifs that have been shown to be involved in beta-cell-specific expression of insulin, Pdx1 and islet amyloid polypeptide (IAPP). In addition, we determined a previously undescribed 5'intron.

Amino Acid Sequence↗
PubMed2000

A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization.

The three-dimensional structure of bacteriophage T7 DNA polymerase reveals the presence of a loop of 4 aa (residues 401-404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family. A genetically altered T7 DNA polymerase, T7 polDelta401-404, lacking these residues, has been characterized biochemically. The polymerase activity of T7 polDelta401-404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3'-to-5' exonuclease activity indistinguishable from that of wild-type T7 DNA polymerase. T7 polDelta401-404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template. T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucleotides synthesized by the primase activity of the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 polDelta401-404 compared with the wild-type enzyme. Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein. The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, presumably, with the T7 gene 4 primase. Gene 4 protein also provides helicase activity for the replication of duplex DNA. T7 polDelta401-404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.

Bacteriophage M13↗
PubMed2000

Valine of the YVDD motif of moloney murine leukemia virus reverse transcriptase: role in the fidelity of DNA synthesis.

The YXDD motif is highly conserved in the reverse transcriptase family. The variable X residue is occupied by valine and methionine in MuLV RT and HIV-1 RT, respectively. Previous studies have shown that Tyr 222, the Y residue of the YXDD motif in MuLV RT, constitutes a major component of the fidelity center of the enzyme [Kaushik, N., Singh, K., Alluru, I., and Modak, M. J. (1999) Biochemistry 38, 2617-2627]. In this work, we present evidence that reverse transcriptases containing valine in the "X" position of the YXDD motif generally catalyze DNA synthesis with greater fidelity than those containing methionine or alanine. In the MuLV RT system, the two mutants V223M and V223A exhibited an overall reduced fidelity of DNA synthesis, specifically for RNA-templated reactions. Further analysis revealed that these mutants exhibit a higher efficiency of misinsertion on MS2 RNA than the wild-type enzyme for every mispair tested. However, unlike HIV-1 RT, the insensitivity of the wild-type MuLV RT to all four ddNTPs remained unchanged by mutation of V223 to Met or Ala. A 3D molecular model of the ternary complex of MuLV RT, template primer, and dNTP suggests that Val 223 along with its neighboring Tyr 222 stabilizes the substrate binding pocket via hydrophobic interactions with the dNTP substrate and template-primer.

Animals↗
PubMed2000

Le Fort I osteotomy and skull base tumors: a pediatric experience.

BACKGROUND: The Le Fort I maxillary osteotomy approach for skull base tumor removal in the pediatric age group has not been widely discussed in the literature except for sporadic case reports and limited case series. OBJECTIVES: To review our experience with the Le Fort I osteotomy and to propose that it be used as an alternative approach because of its many advantages for the removal of tumors of the central skull base and paranasal sinuses. DESIGN: Case series. SETTING: Tertiary academic center. PATIENTS AND METHODS: A 5-year retrospective chart review of cases involving children who had undergone skull base tumor resection via the Le Fort I osteotomy approach. INTERVENTIONS: Skull base tumor removal via the Le Fort I osteotomy approach. MAIN OUTCOME MEASURES: Tumor type, location, and size; intraoperative and postoperative complications; and residual tumor and/or tumor recurrence associated with the surgical approach. RESULTS: Eleven patients (9 boys and 2 girls; mean age, 14.3 years) were identified through the chart review. The tumor types included 8 angiofibromas, 1 malignant fibrous histiocytoma, 1 giant cell tumor, and 1 cavernous hemangioma. All these lesions had extensive tumor growth into at least 1 of the following sites: pterygomaxillary space, sphenoidal sinus, and areas adjacent to the optic nerve, cavernous sinus, clivus, and anterior cranial fossa. The mean follow-up for this cohort was 12.8 months. No intraoperative complications were noted. Postoperative complications were reviewed with respect to the approach. To date, there have been no cases of residual tumor or tumor recurrence that can be attributed to the procedure. CONCLUSIONS: Our experience suggests that the Le Fort I osteotomy approach is a useful technique for the removal of extensive central skull base tumors and paranasal sinuses in the pediatric age group. It has distinct advantages over traditional anterior or lateral approaches, including a more direct line of vision and improved exposure and cosmesis.

Adolescent↗
PubMed2000

Identification of a novel mouse Iroquois homeobox gene, Irx5, and chromosomal localisation of all members of the mouse Iroquois gene family.

The Drosophila genes of the Iroquois-Complex encode homeodomain containing transcription factors that positively regulate the activity of certain proneural Achaete/Scute-C (AS-C) genes during the formation of external sensory organs (J. L. Gomez-Skarmeta and J. Modolell, EMBO J 17:181-190, 1996). Previously, we have identified three highly-related genes of the mouse Iroquois gene family that exert specific expression patterns in the central nervous system (A. Bosse et al., Mech Dev 69:169-181, 1997). In the present paper, we report the identification of a novel member of the Iroquois gene family, Irx5, that shows a restricted spatio/temporal expression during early mouse embryogenesis, distinct from the expression of Irx1-3. An extensive sequence analysis of 20 Iroquois-like genes from seven organisms reveals a high conservation of the homeodomain. Phylogenetic tree reconstruction showed a clustering of the members of the Iroquois gene family into groups of orthologous genes. Together, with the data obtained from the chromosomal mapping analysis, the results indicate that these genes have appeared in vertebrates during evolution as a result of gene duplication.

Animals↗
PubMed2000

Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels.

Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

Alternative Splicing↗
PubMed2000

A new gene trap construct enriching for insertion events near the 5' end of genes.

The gene trap approach is based on the integration of a gene trap vector into the genome. This can be done either by electroporation of a plasmid construct or by infection with a viral vector. Commonly used viral gene trap vectors have been shown to select for integrations near the 5' end of genes. To date, no plasmid vector with a similar tendency has been reported. In this paper we describe a new plasmid vector, pKC199beta geo. This vector contained a short splice acceptor fragment from the Hoxc9 gene, a full length lacZ gene, including an ATG, and a reduced activity, mutant neomycin phosphotransferase gene as a selectable marker. This vector enriched the population of trapped genes in our gene trap screen for insertion events in the 5' end of genes. In the two cases examined the beta-galactosidase activity pattern accurately reflected the endogenous promotor activity.

Animals↗
PubMed2000

Querkopf, a MYST family histone acetyltransferase, is required for normal cerebral cortex development.

In order to find, and mutate, novel genes required for regulation of neurogenesis in the cerebral cortex, we performed a genetic screen in mice. As the result of this screen, we created a new mouse mutant, querkopf. The querkopf mutation is due to an insertion into a MYST family histone acetyltransferase gene. Mice homozygous for the querkopf mutation have craniofacial abnormalities, fail to thrive in the postnatal period and have defects in central nervous system development. The defects in central nervous system development are particularly prominent in the cerebral cortex, which is disproportionally smaller than in wild-type mice. A large reduction in the size of the cortical plate was already apparent during embryogenesis. Homozygous mice show a lack of large pyramidal cells in layer V of the cortex, which is reflected in a reduction in the number of Otx1-positive neurons in this layer during postnatal development. Homozygous mice also show a reduction in the number of GAD67-positive interneurons throughout the cortex. Our results suggest that Querkopf is an essential component of a genetic cascade regulating cell differentiation in the cortex, probably acting in a multiprotein complex regulating chromatin structure during transcription.

Acetyltransferases↗
PubMed1999

The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

Amino Acid Sequence↗
PubMed1999

Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity.

Pax6 is a regulatory gene with restricted expression and essential functions in the developing eye and pancreas and distinct domains of the CNS. In this study we report the identification of three conserved transcription start sites (P0, P1, alpha) in the murine Pax6 locus. Furthermore, using transgenic mouse technology we localized independent cis-regulatory elements controlling the tissue-specific expression of Pax6. Specifically, a 107-bp enhancer and a 1.1-kb sequence within the 4.6-kb untranslated region upstream of exon 0 are required to mediate Pax6 expression in the lens, cornea, lacrimal gland, conjunctiva, or pancreas, respectively. Another 530-bp enhancer fragment located downstream of the Pax6 translational start site is required for expression in the neural retina, the pigment layer of the retina, and the iris. Finally, a 5-kb fragment located between the promoters P0 and P1 can mediate expression into the dorsal telencephalon, the hindbrain, and the spinal cord. The identified Pax6/cis-essential elements are highly conserved in pufferfish, mouse, and human DNA and contain binding sites for several transcription factors indicative of the cascade of control events. Corresponding regulatory elements from pufferfish are able to mimic the reporter expression in transgenic mice. Thus, the results indicate a structural and functional conservation of the Pax6 regulatory elements in the vertebrate genome.

Animals↗
PubMed1999

Prox1 function is crucial for mouse lens-fibre elongation.

Although insights have emerged regarding genes controlling the early stages of eye formation, little is known about lens-fibre differentiation and elongation. The expression pattern of the Prox1 homeobox gene suggests it has a role in a variety of embryonic tissues, including lens. To analyse the requirement for Prox1 during mammalian development, we inactivated the locus in mice. Homozygous Prox1-null mice die at mid-gestation from multiple developmental defects; here we describe the specific effect on lens development. Prox1 inactivation causes abnormal cellular proliferation, downregulated expression of the cell-cycle inhibitors Cdkn1b (also known as p27KIP1) and Cdkn1c (also known as p57KIP2), misexpression of E-cadherin and inappropriate apoptosis. Consequently, mutant lens cells fail to polarize and elongate properly, resulting in a hollow lens. Our data provide evidence that the progression of terminal fibre differentiation and elongation is dependent on Prox1 activity during lens development.

Animals↗
PubMed1999

Primary amyloidosis presenting as massive cervical lymphadenopathy with severe dyspnea: a case report and review of the literature.

Amyloidosis is an end point of several diseases in which the unifying characteristic is the association and extracellular deposition of the insoluble protein fibrils within various tissues and organs. We have discussed a variety of clinical findings of primary amyloidosis with emphasis on its otolaryngologic manifestations. This case represents the second report in the literature of a neck mass resulting from a plasma cell dyscrasia and the first report of AL disease producing such extensive cervical lymphadenopathy as to cause significant respiratory compromise.

Aged↗
PubMed1999

Juvenile nasopharyngeal angiofibroma.

This is a retrospective study of 17 patients with juvenile nasopharyngeal angiofibroma treated from 1983 to 1996. Patients with Stage I or II disease according to the Fisch classification system were treated surgically by a transpalatal approach. One patient underwent a Le Fort I osteotomy and down-fracture approach for access. Three patients underwent combined transpalatal and lateral rhinotomy for access, whereas one underwent a transcervical double mandibular osteotomy to facilitate the exposure. A patient with Stage IV disease underwent a combined subcranial frontonasal osteotomy plus a Le Fort I osteotomy for access to a massive angiofibroma. Initial surgical management prevented recurrence in 79% of patients. Two patients with intracranial extension were treated with primary irradiation therapy; their tumours became asymptomatic. Preoperative angiography and embolization were used to treat all surgical candidates. The use of newer craniofacial or subcranial techniques and infratemporal fossa approaches with osteotomies can provide access to large angiofibromas even when there is skull base or intracranial involvement. Surgical exposure may also be enhanced by the use of the Le Fort I osteotomy and down-fracture approaches.

Adolescent↗
PubMed1998

Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection.

We have used different gene trap vectors and in vitro preselection of embryonic stem (ES) cells for a large scale screening of insertional mutations in developmentally regulated genes. A gene trap vector was constructed, which contains an internal ribosome entry site (IRES) upstream from a betageo selectable-reporter fusion gene. Analysis of 801 independent integrations revealed that the IRESbetageo vector allows for a global enrichment of about 15 folds in the number of detectable gene trap events when compared with a conventional betageo vector. Characterization of in vitro and in vivo lacZ expression suggested that this IRES-based vector is able to capture a wide range of genes expressed in a variety of tissues and developmental stages, and it can also allow trapping of genes expressed at very low levels in ES cells. A preselection protocol was devised, where gene-trapped ES cells were grown in the presence of specific growth/differentiation factors such as follistatin, nerve growth factor, and retinoic acid. Several gene trap integrations were found to be either activated or repressed by one of these factors. Characterization of lacZ expression during embryogenesis showed a strong enrichment of restricted patterns in vivo after ES cell preselection. These results suggest that a combination of IRESbetageo vector and in vitro preselection is more effective for the capture and mutation of a large number of developmental genes.

Animals↗
PubMed1998

Genes expressed after retinoic acid-mediated differentiation of embryoid bodies are likely to be expressed during embryo development.

In order to test if retinoic acid-mediated differentiation of embryoid bodies can be used as an in vitro preselection method for ES cell lines generated by gene trap, we correlated gene expression after in vitro differentiation and in 11.5-day embryos. Fifty-two genes captured by gene trap and expressed in undifferentiated embryonic stem cells were analyzed. Most genes expressed after differentiation in vitro were also expressed during embryo development. In order to correlate the expression patterns in vitro and in vivo, the in vitro expression in the center and in the periphery of the embryoid body outgrowths was observed. This allowed us to distinguish, according to in vitro expression, not expressed genes from those expressed widely in 11.5-day embryos. Consequently, with this parameter we increased the probability to obtain the restricted expression patterns in vivo. This study demonstrates the potential of the differentiation procedure in combination with the gene trap to select in vitro for genes expressed during embryo development.

Animals↗
PubMed1998

Gene trap expression and mutational analysis for genes involved in the development of the mammalian nervous system.

We have used a large-scale gene trap approach for the isolation and mutation of genes that might play roles in the developing nervous system. After in vitro integration of two different gene trap vectors (pGT1.8geo: Skarnes et al. [1995] Proc. Natl. Acad. Sci. USA 92:6592-6596; IRES beta geo: Chowdhury et al. [1997] Nucleic Acids Res. 25:1531-1536) in mouse embryonic stem (ES) cell lines, we created 64 transgenic mouse lines. The expression analysis of the reporter gene during embryogenesis of heterozygous embryos revealed 47 lines with a variety of patterns. Around one-third (36%) of these gene trap lines showed spatiotemporal expression that was either restricted predominantly in the developing nervous system (11 lines; 17%) or widespread but with very high levels of expression in the nervous tissue (12 lines; 19%). In most cases, a correlation was found between the in vitro and the in vivo patterns of the reporter gene expression. Thus far, preliminary mutant analysis of 16 gene trap lines with potentially interesting expression patterns in the developing nervous system showed that mice homozygous for eight (50%) insertions were lethal, whereas the homozygous mice from five gene trap lines (31%) showed a lower than expected Mendelian ratio of live homozygous animals. Analysis of beta-galactosidase reporter gene expression during embryogenesis has shown that four transgenic lines are useful lacZ in situ markers for specific regions of the developing nervous system. Here, we discuss some in vivo and in vitro selection criteria that may increase the number of the trapped genes potentially involved in the control of neural development and some future strategies to improve further the efficiency of the gene trap approach.

Animals↗