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K Mishra

Publications and source records attributed to K Mishra.

29 records · Page 2Linked to original sources
PubMed1995

Essential role of protein phosphorylation in nuclear transport.

We have investigated a possible role for protein phosphorylation in nuclear transport in semi-intact cells, prepared by digitonin permeabilization of rat F-111 fibroblasts. Treatment of semi-intact cells with alkaline phosphatase abolished the import of nuclear transport substrates, namely, signal peptide-albumin conjugates, as well as their signal-dependent binding at the nuclear pores, but did not affect the morphology of the cells, in particular their cytoskeletal network. Authentic transport and functional binding of the karyophilic protein at the nuclear envelope could be restored by incubation of phosphatase-treated cells with cytosol enriched in protein kinase C or with purified protein kinase A (catalytic subunit). Restoration of transport was blocked by specific inhibitors of these kinases. Since the protein phosphorylation required for nuclear transport appeared to be a reasonably stable modification, characterization of the phosphorylated proteins was attempted in kinase reactions with radiolabeled ATP. Two proteins of 60-62 kDa were the predominant substrates phosphorylated by both protein kinase C and protein kinase A under conditions wherein nuclear transport was restored. Our results suggest a requirement for phosphorylation of one or more proteins for binding of a karyophilic protein at the nuclear envelope.

Albumins↗
PubMed1994

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins.

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.

Amino Acid Sequence↗
PubMed1994

Developmentally regulated Gb3 galactosyltransferase and alpha-galactosidase determine Shiga toxin receptors in intestine.

The receptor for Shiga toxin on rabbit intestinal microvillus membranes (MVMs) has been identified as a developmentally regulated glycolipid, globotriaosylceramide [galactose alpha 1-4 galactose beta 1-4 glucose beta 1-1 ceramide (Gb3)]. MVM Gb3 levels increase markedly in the third week of life, concomitant with fluid secretory responses to the toxin. To study mechanisms controlling developmental regulation of MVM Gb3, we measured the specific synthetic Gb3 galactosyltransferase and degradative alpha-galactosidase activities and subcellular distribution of Gb3 at various ages. Quantitative high-performance liquid chromatography demonstrated a similar developmental pattern in both microsomal and MVM Gb3, indicating that late expression of MVM Gb3 is not due to delayed migration of Gb3 from the microsomal to the MVM. The specific Gb3 galactosyltransferase activity increased with age, with a sharp increase seen at 18 days of age, whereas alpha-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age. The nature of the signals for developmental regulation of Gb3 levels is unknown, as are the physiological consequences of altered MVM glycolipid composition other than mediating response to Shiga toxin.

Aging↗
PubMed1994

A conserved epitope on nuclear pore phosphoproteins reflects cell division status.

The nuclear pore complexes mediate the selective nuclear import of proteins in a signal- and energy-dependent process. We have earlier reported the characterization of a monoclonal antibody, Mab E2, that recognizes a novel class of nuclear pore phosphoproteins involved in signal-binding and protein transport. In the present study, we have analyzed the pattern of immunoreactivity of Mab E2 in cultured rat fibroblasts and have observed significant differences in the expression of epitopes in proliferating and quiescent cells. Furthermore, the common epitope recognized by Mab E2 is conserved across species, consistent with its essential role in nuclear protein import.

Amino Acid Sequence↗
PubMed1993

A quantitative immunostaining method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase for the synthesis of globotriaosylceramide in rabbit small intestine and HeLa cells.

Galactosyltransferase is required for the addition of galactose to lactosylceramide (galactose beta 1-4 glucose beta 1-1 ceramide), resulting in the synthesis of globotriaosylceramide (Gb3). We describe a quantitative more sensitive and specific method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase activities in rabbit small intestine and HeLa cell which utilizes the specific binding of Shiga toxin to the product, Gb3. Intestinal microsomal or HeLa cell sonicate preparations were incubated in the presence of lactosylceramide and [14C]UDP-galactose. The lipid reaction products were extracted on C18 Bond-Elut columns, separated by high-performance thin-layer chromatography and exposed to Shiga toxin followed by polyclonal rabbit anti-Shiga toxin antibody and goat anti-rabbit IgG alkaline phosphatase conjugate. Gb3 was visualized with NBT and BCIP and quantitated by densitometry. These data were compared with a standard assay in which, following incubation and lipid extraction, radioactivity was measured by scintillation counting of the isolated lipids. There was a 22-fold increase in enzyme activity by the immunostaining method compared to the usual scintillation counting technique. This is attributable to the exclusion of radioactive lipids other than Gb3 in calculating enzyme activity and the correction for endogenous UDP-galactose. Thus, the immunostaining method provides increased accuracy, sensitivity, and specificity in the assay of galactosyltransferase activity.

Animals↗
PubMed1986

Effect of early iron deficiency in rat on the gamma-aminobutyric acid shunt in brain.

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in gamma-aminobutyric acid (GABA) (13%, p less than 0.01) and glutamic acid (20%, p less than 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA-transaminase, and of the NAD+-linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+-linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron-supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.

4-Aminobutyrate Transaminase↗
PubMedDate not supplied

Congenital spinal lipomas--tumor versus hamartoma.

Congenital spinal lipomas as a form of dysraphism are frequently reported in literature. A few studies have highlighted their histologic features and found them different from lipomas in other parts of the body. In a review of eight cases of spinal lipomas, we found heterotopic tissue in seven. Neural tissue, muscle, bone and cartilage were frequently encountered. The presence of meningothelial cells in one case was an unusual finding. These findings support the concept of their hamartomatous origin. Malignancy, occurring later in life in one of the heterotopic elements is known, though uncommon, and is one of the reasons, besides neurologic deficit, for long term follow-up.

Child, Preschool↗
PubMedDate not supplied

Fine needle aspiration cytology of malignant chondroid syringoma: a case report.

BACKGROUND: Chondroid syringoma, a tumor of the eccrine glands, was previously called mixed tumor of skin as it has both mesenchymal and epithelial elements. Malignancy in this tumor is extremely rare. Although there are a few reports describing the cytomorphologic features of chondroid syringoma, the cytologic findings of its malignant counterpart have not been described. CASE: A 40-year-old female presented with a recurrent swelling on the scalp of one year's duration. Fine needle aspiration yielded blood-mixed gelatinous material. May-Grünwald-Giemsastained smears showed epithelial cells arranged in cordlike structures and ill-formed glands against a myxomatous background. The epithelial cells had scanty cytoplasm and markedly pleomorphic nuclei with prominent nucleoli. A few cells in the stroma had a halo around them and a resemblance to cartilage cells. A preoperative diagnosis of malignant chondroid syringoma was made. The tumor was excised, and the cytologic diagnosis was confirmed on histopathology. CONCLUSION: Cytomorphologic features of a rare case of malignant chondroid syringoma are reported for the first time. The presence of malignant epithelial cells against a myxoid background with a few chondroid foci helped in making a correct preoperative diagnosis.

Adenoma, Pleomorphic↗
PubMedDate not supplied

Do all grade I lesions on colposcopy need to be biopsied?

The present study intends to correlate grade I atypical transformation zone (ATZ) on colposcopy with cytology & histology in 51 patients by retrospective data analysis. Indications of colposcopy were inflammatory smears with unhealthy cervix (37/51). Atypical squamous cells of unknown etiology (2/51) & squamous intraepithelial lesions on cytology (12/51). All the patients exhibited grade I lesions on colposcopy & underwent directed biopsy. On histology chronic cervicitis was the commonest finding 70.6% (36/51) & CIN was found in 19.6% (10/51), out of which CIN II-III occurred only in 3.92% (2/51). Overcall rate of colposcopy for grade I lesions was 80.39%. Age, parity, the postcoital or contact bleeding did not correlate with the histological positivity of the lesions. Grade I ATZ with inflammatory smears revealed CIN II-III only in 2.7% (1/37) while with low grade SIL cytology there was no moderate or severe dysplasia. However Grade I lesions in association with high grade SIL exhibited CIN II-III lesions in 16.6% (1/6). Therefore grade I lesions in presence of inflammatory or low grade SIL smears can be observed & biopsied only if the changes persist. However association of high grade SIL with grade I ATZ calls for immediate biopsy.

Adolescent↗