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G Srivastava

Publications and source records attributed to G Srivastava.

At least 19 recordsLinked to original sources
PubMed1992

Enzymatic transfer of a preassembled trisaccharide antigen to cell surfaces using a fucosyltransferase.

The Lewis alpha (1-->3/4)-fucosyltransferase (Le-FucT) is known to fucosylate both Type I (beta Gal(1-->3) beta GlcNAc) and Type II (beta Gal(1-->4) beta GlcNAc) sequences even when these are sialylated at OH-3 or fucosylated at OH-2 of the terminal Gal residues. These acceptor sequences are ubiquitous on mammalian cell-surface glycoproteins and glycolipids. The Le-FucT enzyme is therefore a potential candidate as a universal reagent for the modification of cell surfaces. We have found that a readily accessible, partially purified Le-FucT from human milk, which normally uses GDP-fucose (a 6-deoxy sugar) as the donor for the transfer of a single fucose residue, will also transfer a fucose residue substituted on C-6 by a very large sterically demanding structure, in this instance, a synthetic blood group antigen. As a demonstration of the ability of the Le-FucT to modify glycoconjugates in a mild and specific manner, we chemically synthesized the complex sugar-nucleotide alpha Gal(1-->3) [alpha Fuc(1-->2)]-beta Gal-O-(CH2)8COHN(6)-beta-L-fucose-GDP (13) which is a GDP-fucose analog where the human blood group B trisaccharide antigen is covalently linked to C-6 of fucose through an amino group. It is shown that, in enzyme-linked immunosorbent assays, the Le-FucT uses both immobilized beta Gal(1-->3) beta GlcNAc-bovine serum albumin conjugates and fetuin as acceptor substrates and renders them blood group B-active as detected by a monoclonal anti-B blood-grouping antibody. The fucose residue to which the B-trisaccharide is linked therefore becomes covalently attached to the acceptor oligosaccharide chains of those glycoproteins. Incubation of type "O" erythrocytes with the Le-FucT and complex donor 13 results in the covalent transfer of alpha Gal(1-->3) [alpha Fuc(1-->2)] beta Gal-O-(CH2)8COHN(6)-beta-L-Fuc to cell-surface acceptors since the cells become phenotypically "B" and are agglutinated by the same antibody. It is proposed that the Le-FucT represents a powerful new tool with the ability to label animal cell surfaces with preassembled oligosaccharide and possibly also other complex recognition markers.

ABO Blood-Group System↗
PubMed1992

Synthesis of a tetrasaccharide acceptor for use in the assay of UDP-GlcpNAc:beta-D-Galp-(1----4)-beta-D-GlcpNAc (GlcNAc to Gal) beta(1----3)-N-acetylglucosaminyltransferase activity and the pentasaccharide product that would be formed by its enzymic glycosylation.

The tetrasaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-alpha-D- Manp-(1----6)-beta-D-Manp-OR (2) and the pentasaccharide beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp-OR (3), where R = (CH2)8COOMe, have been prepared by using combined chemical and enzymic procedures. Structure 2 is a substrate for UDP-GlcpNAc:beta-D-Galp-(1----4)- beta-D-GlcpNAc (GlcNAc to Gal) beta(1----3)-N-acetylglucosaminyltransferase, and 3 would be the product of its action. Antibodies raised against 3 are intended for use in an ELISA assay that would quantitate the enzymic conversion of immobilized 2 into 3.

Amino Sugars↗
PubMed1992

Effect of dexamethasone on mRNA levels for 5-aminolevulinate synthase in different rat tissues.

5-Aminolevulinate synthase mRNA levels from different tissues were quantitated by Northern blot hybridization analysis utilizing the rat liver 5-aminolevulinate synthase cDNA clone as probe. A 5-aminolevulinate synthase mRNA species of size 2.3 kb was seen in all the tissues examined. Densitometric scanning of the autoradiographs demonstrated that the adrenal gland contained the largest amount of 5-aminolevulinate synthase mRNA. Levels corresponding to approximately 50% of this amount were found in the small intestine, lung, heart, muscle and testes. In the liver and kidney the level was approximately 25% of that found in the adrenal gland. These results demonstrate the housekeeping role of this gene. Dexamethasone treatment for 1 day or 5 days dramatically induced 5-aminolevulinate synthase mRNA levels in the liver and small intestine, and to a lesser extent in lung, heart, kidney and muscle. Nuclear run-off experiments suggest that a post-transcriptional mechanism predominantly contributes to the dexamethasone-induced increase in 5-aminolevulinate synthase mRNA levels observed in the liver. Interestingly, in the steroidogenic tissues of the adrenal gland and testes, there was a substantial decrease in 5-aminolevulinate synthase mRNA levels after dexamethasone administration but the mechanism of this control remains to be investigated.

5-Aminolevulinate Synthetase↗
PubMed1992

Human papillomavirus and its prognostic significance in invasive carcinoma of the cervix in young patients.

Of 1,200 Chinese patients treated for carcinoma of the cervix between 1975 and 1984, those aged 40 years (n = 70) or less had a poorer prognosis. Of the latter group, 71% had disease classified as less than International Federation of Gynecology and Obstetrics (FIGO) stage Ib, and in 91% the disease was less than stage IIa; however, the 5-year survival of the 45 patients with verified invasive carcinoma was 75% compared with 82% for the overall group. The polymerase chain reaction (PCR) was used to test for the presence of human papilloma virus (HPV) types 16 and 18 within paraffin-embedded tissue from the primary tumors. HPV-16 DNA sequences were detected in 69% of the cases, HPV-18 DNA sequences in 44%, and in 31% both HPV-16 and -18 DNA could be identified. Of the cases of invasive carcinoma, 82% (37 of 45) contained DNA of HPV types 16 and/or 18. The effects of the presence or absence of HPV DNA on known prognostic factors was investigated. Although the influence of traditional factors, especially lymph node involvement, was confirmed, careful statistical analysis could not demonstrate a prognostic influence for HPV-16/18.

Adult↗
PubMed1992

Deletions, duplications and novel restriction fragment length polymorphism in Duchenne and Becker muscular dystrophies.

To determine the mutations of Southern Chinese with Duchenne and Becker muscular dystrophies (DMD, BMD), we analysed 28 DMD and BMD patients in 24 unrelated families for intragenic deletions and duplications by using cDNA probes covering the entire 14 kb of the dystrophin gene. Deletions were detected in nine unrelated patients (seven patients by probe 8 and two by probe 2b-3). Gene duplications were detected by probe 1-2a in two patients with the duplication bands confirmed in both Hind III and Bgl II digests and by densitometry. A third patient was found to have a junction fragment with Bgl II and a duplication band with Hind III by probe 5b-7. Therefore 50% of the 24 unrelated families were found to have either deletions or duplications. A previously undescribed restriction fragment length polymorphism (RFLP) was found in one family with probe 5b-7 in Bgl II digests which was found to segregate with the disease phenotype. This new RFLP was not detected in over 70 unrelated X chromosomes we have examined so far, and appeared to be "private" for this family. The presence of this new restriction site may or may not be the mutation responsible for the disease phenotype.

Asian People↗
PubMed1992

Human papillomavirus 16/18 and nasopharyngeal carcinoma.

Sixteen cases of nasopharyngeal carcinoma (eight anaplastic and eight well differentiated squamous types) were examined for the presence of human papillomavirus types 16 and 18 genomes using the polymerase chain reaction on paraffin wax embedded biopsy specimens. Although nasopharyngeal carcinoma, particularly the anaplastic type, is strongly associated with Epstein-Barr virus, other factors may be involved in its pathogenesis. No DNA of either human papillomavirus subtype was detected. It is concluded, therefore, that these two "high risk" types of human papillomavirus are not implicated in the pathogenesis of nasopharyngeal carcinoma. The number of cases in this series was small, however, and further studies are warranted using fresh biopsy material and including other viral subtypes.

Base Sequence↗
PubMed1992

Epstein-Barr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong.

AIMS: To investigate the presence of Epstein-Barr virus (EBV) in cases of nasopharyngeal carcinoma (NPC) in Chinese patients living in Hong Kong. METHODS: Nasopharyngeal biopsy specimens, formalin fixed and paraffin wax embedded, from 24 patients, eight with undifferentiated nasopharyngeal carcinoma, eight with well differentiated squamous carcinoma, and eight showing normal tissue histology, were analysed for the presence of Epstein-Barr virus (EBV) DNA by slot-blot hybridisation on extracted unamplified DNA, and also after amplification of EBV specific sequences by the polymerase chain reaction (PCR). RESULTS: DNA slot-blot analysis showed viral DNA in all the undifferentiated, five of the well differentiated tumours, and none of the normal biopsy specimens. PCR studies confirmed positivity in the eight undifferentiated tumours, but six of the well differentiated tumours and three of the normal biopsy specimens showed viral DNA by this method, illustrating its greater sensitivity. CONCLUSIONS: EBV genome is present in appreciable copy number in most cases of well differentiated NPC in Chinese patients in Hong Kong.

Base Sequence↗
PubMed1992

Divergent effects of glucocorticoid on the gene expression of vasoactive intestinal peptide in the rat cerebral cortex and pituitary.

We investigated the effects of glucocorticoid on the expression of the vasoactive intestinal peptide (VIP) gene, a neuropeptide and an established prolactin (PRL)-releasing factor, in the rat brain and pituitary. The mRNA and peptide contents of VIP in the cerebral cortex, hypothalamus and anterior pituitary of male Sprague-Dawley rats were quantitated 4 weeks after adrenalectomy or sham-operation. Following adrenalectomy, VIP mRNA content increased in the anterior pituitary but showed no significant change in the cerebral cortex and hypothalamus. Dexamethasone treatment for 10 days abolished the effect of adrenalectomy and decreased significantly pituitary VIP mRNA content in sham-operated rats. In the cerebral cortex, however, dexamethasone treatment resulted in an enhancement in VIP mRNA levels in both sham-operated and adrenalectomized animals. Hypothalamic VIP mRNA content remained unchanged. These changes in VIP mRNA levels were accompanied by parallel changes in VIP concentrations in the tissues studied, suggesting that glucocorticoid regulates the synthesis of VIP in the cerebral cortex and anterior pituitary. On the other hand, serum PRL level increased after adrenalectomy but became suppressed following dexamethasone administration, in parallel with changes in pituitary VIP synthesis. These findings suggest that the effect of glucocorticoid on PRL secretion may be mediated, at least in part, via changes in VIP synthesis and secretion. We conclude that glucocorticoid regulates the expression of VIP in the rat brain, resulting in divergent changes in the cerebral cortex and pituitary. Changes in VIP synthesis and secretion may contribute to the disturbances in brain function and PRL secretion in conditions of glucocorticoid excess.

Adrenalectomy↗
PubMed1992

Clonal Epstein-Barr virus genome in T-cell-rich lymphomas of B or probable B lineage.

Seventeen nodal lymphomas (originally diagnosed as T-cell lymphomas based on histological features and immunohistochemical staining results) were studied for the presence of Epstein-Barr virus (EBV) genome, and the results correlated with immunoglobulin and T-cell receptor gene rearrangement analyses performed on the same tissue samples. All four EBV positive cases had clonal rearrangement of the joining region of the immunoglobulin heavy chain (IgJH) gene without clonal T-cell receptor beta-chain (TCR beta) gene rearrangement. Of these, two cases also showed clonally rearranged light chain gene, and they were reclassified as T-cell rich B-cell lymphomas (TRBL). The other two cases lacked clonal kappa or lambda light chain rearrangement and they were reclassified as T-cell rich lymphomas of probable B lineage, based on their isolated IgJH clonal rearrangement. These B-cell lymphomas may be easily misdiagnosed as T-cell lymphomas owing to the presence of an abundant reactive T-cell infiltrate masking the tumor population. The florid T-cell reaction may represent an unusual host response towards a clonal proliferation of EBV bearing B cells.

Blotting, Southern↗
PubMed1992

Detection of clonal Epstein-Barr virus in malignant proliferation of peripheral blood CD3+ CD8+ T cells.

Epstein Barr virus (EBV) DNA was detected in a monoclonal proliferation of T cells in a three-year-old girl who presented with a history of fever, hepatosplenomegaly, and generalised lymphadenopathy. The disease ran a rapid, fulminant course and the patient died 11 days after presentation. Examination of the blood showed a lymphocytosis of 50 x 10(9)/l with all the cells showing the morphology of large granular lymphocytes. These cells were CD2+3+8+25+. Cytogenetic studies showed the presence of a 6q- clone. Southern blotting and hybridisation with a constant region probe for the T-cell receptor (TCR) beta chain gene showed clonal rearrangement of the TCR beta gene. Hybridisation of the Southern blot to the EBV XhoI probe revealed a clonal pattern of episomal EBV DNA. Our results establish the association between clonal EBV infection to a malignant proliferation of peripheral blood CD8+ T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases.

The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.

Animals↗
PubMed1991

Human papillomavirus 6, 11, and 16 in laryngeal papillomas.

Twenty-seven cases of benign laryngeal papillomas, both single and multiple variants, were analysed for human papillomavirus (HPV) by DNA slot-blot hybridization chiefly to determine the pattern of infection in Hong Kong Chinese. DNA was extracted from paraffin blocks of formalin-fixed tissue and probed separately for HPV 6, 11, 16, and 18. Sixteen cases (59 per cent) showed the presence of at least one of these four HPV genomes. Thirteen cases (48 per cent) were positive for HPV 11 only. Three other cases (11 per cent) showed triple positivity for HPV 6, 11, and 16. None were positive for HPV 18. The predominance of HPV 11 infection contrasts with other series which have shown either an almost equal distribution of HPV 6 and 11 or a predominance of HPV 6. The finding of HPV 16 in three cases was unexpected. Using the polymerase chain reaction (PCR) with primers complementary to the upstream regulatory region of the HPV 16 viral DNA, the presence of HPV 16 genome was confirmed in all three cases. As the number of HPV 16-positive cases in this study is small, analysis of more cases using fresh biopsy material and a wider range of HPV type-specific PCR primers is warranted to determine the relative incidence of HPV subtypes in these benign laryngeal papillomas.

Adolescent↗
PubMed1991

Prevalence of low and high risk human papillomavirus types in cervical cells from Hong Kong pregnant Chinese using filter in situ hybridization.

Filter in situ hybridization, using separate probes for human papillomavirus deoxyribonucleic acid types 6, 11, 16 and 18, was used to determine the prevailing HPV types amongst a group of pregnant Chinese women. This group had been previously identified using a mixture of probes for HPV types 6, 11, 16, 18, 31, 33, 35 (Virapap). Specimens from six of the eleven cases contained one HPV type (55%) while five were positive for two types (45%). The "low-risk" types (HBV6, 11) were identified as frequently as the "high-risk" types (HPV16, 18) in this study.

Cross-Sectional Studies↗
PubMed1990

Combined chemical-enzymic synthesis of deoxygenated oligosaccharide analogs: transfer of deoxygenated D-GlcpNAc residues from their UDP-GlcpNAc derivatives using N-acetylglucosaminyltransferase I.

The 3''-, 4''-, and 6''-deoxy analogs of UDP-GlcpNAc have been synthesized chemically and found to act as donor-substrates for N-acetylglucosaminyltransferase-I (GnT-I) from human milk. Incubation of UDP-GlcpNAc and these deoxy analogs with GnT-I in the presence of alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -O(CH2)8COOMe gave beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp- (1----6)]- beta-D-Manp-O(CH2)8COOMe (6), and the deoxy analogs 12-14 where HO-3, HO-4, and HO-6, respectively, of the beta-D-GlcNAc residue were replaced by hydrogen. The tetrasaccharide glycosides 6 and 12-14 were characterized by 1H-n.m.r. spectroscopy and evaluated as acceptors for GnT-II, the next enzyme in the pathway of biosynthesis of Asn-linked oligosaccharides. Deoxygenation of the 3-position of the beta-D-GlcNAc residue of 6 completely abolished its acceptor activity, whereas removal of HO-4 or HO-6 caused only modest decreases in activity.

Acetylglucosamine↗
PubMed1990

Sex-related differences and thyroid hormone regulation of vasoactive intestinal peptide gene expression in the rat brain and pituitary.

To determine the effects of hypothyroidism and sexual dimorphism on the gene expression of vasoactive intestinal peptide (VIP) in the rat brain, VIP mRNA levels were measured in the cerebral cortex, hypothalamus and anterior pituitary of control and hypothyroid (thionamide-treated) rats. A tissue-specific increase in VIP mRNA level was observed in the hypothyroid pituitary. In addition, higher levels of VIP mRNA were found in the pituitaries of male rats suggesting a sex-related difference in VIP gene expression. Similar differences were not observed in the cortex or hypothalamus.

Animals↗
PubMed1990

Immunocytochemical studies on the localization of 5-aminolevulinate synthase in rat liver.

The localization of 5-aminolevulinate synthase (ALAS) in hepatocytes of untreated and porphyrinogenic drug-treated rats has been examined by an immunocytochemical approach using a monoclonal antibody and protein A-gold labeling. Gold particles representing antigenic sites for ALAS were observed in the mitochondria and cytoplasm of untreated and drug-treated cells. Quantitative analysis of the labeling density showed that levels of ALAS increased significantly in both of these cellular compartments following drug treatment. Evidence that the detected cytoplasmic form of ALAS represents the precursor of the enzyme was obtained from immunoblotting experiments. The direct detection of cytosolic ALAS in vivo rules out the possibility that enzyme activity previously detected in the cytosol fraction resulted from mitochondrial leakage during cell fractionation. The results indicate that the cytosolic accumulation of ALAS is not a consequence of the inability of mitochondria to accommodate more enzyme. However, the molecular basis for this cytosolic accumulation is not known. The studies also established that the mitochondrial enzyme is predominantly, if not exclusively, associated with the matrix side of the inner mitochondrial membrane.

5-Aminolevulinate Synthetase↗
PubMed1990

Translocation of bcl-2 gene in non-Hodgkin's lymphomas in Hong Kong Chinese.

The close association between translocation of the proto-oncogene bcl-2 and follicular lymphomas has been well established in Caucasian patients and the de-regulation of bcl-2 has been implicated in follicular lymphomagenesis. Similar molecular structural alterations have also been detected in diffuse lymphomas with a previous history of a follicular pattern as well as in a smaller proportion of de novo diffuse lymphomas. There is a lower incidence of follicular lymphomas in Chinese. In order to investigate further this phenomenon, we used bcl-2 translocation as a genetic marker of follicular lymphomas, to study 31 cases of B cell non-Hodgkin's lymphomas in Chinese patients by Southern blot analysis. Eight out of 16 cases of follicular lymphomas showed bcl-2 translocation with involvement of the major breakpoint region (MBR). Six of these cases utilized breakpoints within the 4.3 kb HindIII fragment, while in two cases the breakpoints were more dispersed, but still within the BamHI fragment. An additional case of follicular lymphoma showed translocation of bcl-2 gene with involvement of the minor cluster region (mcr), making a total of nine out of 16. None of the 15 cases of diffuse lymphomas showed similar molecular structural alterations. These data show that bcl-2 translocation is present in 57% of follicular lymphomas in Chinese patients, and support the notion that bcl-2 translocation is a consistent marker for follicular lymphomas irrespective of ethnic differences. As the translocation is not detected in the diffuse lymphomas, there is no evidence to suggest that the low incidence of follicular lymphomas in Chinese patients is due to a greater tendency for follicular tumours to progress rapidly and present as diffuse lymphomas.

Blotting, Southern↗