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K Patel

Publications and source records attributed to K Patel.

At least 235 records · Page 13Linked to original sources
PubMed1990

Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone.

Deamidation of Asn residues is one of the major chemical pathways of degradation of proteins and peptides. Adrenocorticotropic hormone (ACTH), a 39-amino acid polypeptide with a single Asn residue, was shown in this study to be a useful model polypeptide for the study of the effects of pH and buffer concentration on the rate and pathway of deamidation. The disappearance of ACTH and appearance of deamidated ACTH were monitored by isoelectric focusing (IEF), and ammonia production was monitored spectrophotometrically using a coupled enzymatic assay. Using these analytical methods, the deamidation of ACTH was shown to follow pseudo-first-order kinetics and was dependent on pH and buffer concentrations. The separation of the deamidated ACTHs (Asp-ACTH and isoAsp-ACTH) from ACTH was successful, but attempts to separate Asp-ACTH from isoAsp-ACTH using cation-exchange HPLC and IEF were unsuccessful. Using bovine protein carboxymethyltransferase (PCM), which selectively methylates the carboxyl group of isoAsp residue, the isoAsp-ACTH could be detected at pH 7.0 and 9.6 but not at pH 1.9. These data support the hypothesis that under neutral and alkaline conditions, deamidation of ACTH proceeds through the formation of a cyclic imide intermediate (slow step), followed by its hydrolysis to the Asp-ACTH and isoAsp-ACTH (fast step). Under acidic conditions, the reaction appears to proceed via direct hydrolysis of the Asn residue to form Asp-ACTH without the formation of a cyclic imide intermediate.

Adrenocorticotropic Hormone↗
PubMed1990

Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides.

Deamidation of Asn residues can occur either by direct hydrolysis of the Asn residue or via a cyclic imide intermediate. The effects of primary sequence on the pathways of deamidation of Asn residues were studied using Val-Tyr-X-Asn-Y-Ala hexapeptides with substitution on the C-terminal side (Y) and on the N-terminal side (X) of the Asn residue. In acidic media the peptides deamidate by direct hydrolysis of the Asn residue to yield only Asp peptides, whereas under neutral or alkaline conditions, the peptides deamidate by formation of the cyclic imide intermediates which hydrolyze to yield both isoAsp and Asp peptides. At neutral to alkaline pH's the rate of deamidation was significantly affected by the size of the amino acid on the C-terminal side of the Asn residue. The amino acid on the C-terminal side of the Asn residue has no effect on the rate of deamidation at acidic pH. Changes in the structure of the amino acid on the N-terminal side of the Asn residue had no significant effect on the rate of deamidation at all the pH's studied. For peptides that underwent deamidation slowly, a reaction involving the attack of the Asn side chain on the peptide carbonyl carbon resulting in peptide bond cleavage was also observed.

Amino Acid Sequence↗
PubMed1990

Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript.

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells, promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern (RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor of the more stable 2- and 1.3-kb LATs.

Animals↗
PubMed1990

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.

Adsorption↗
PubMed1990

Immunomagnetic manipulation of bone marrow and tumour cells: an update.

The immunomagnetic separation procedure for the separation of neuroblasts from bone marrow harvested for autologous transplantation was first described in 1983 (Treleaven et al., 1984). In the intervening period, the technique has been extended to other tumours and modified by several laboratories. In addition, the procedure has been used for the separation of different mammalian cell types (Nilsson et al., 1987), micro-organisms (Lund et al., 1988) and at the subcellular level, organelles (Howell et al., 1988), and DNA (Dudin et al., 1988). Although immunomagnetic separation techniques have been used extensively for bone marrow purging, the recovery of cells from the matrix has remained problematical. This manuscript is intended to offer an update on our experiences in the use of the immunomagnetic purging procedure. In addition, more detailed basic studies have been undertaken to characterize further the antigens recognized by the antibodies for the removal of neuroblasts from bone marrow. The information obtained in these studies may, in part, explain why it is intrinsically more difficult to separate neuroblasts from the magnetic matrix than haematopoietic cells such as either T or acute lymphoblastic leukaemic cells.

Antibodies, Monoclonal↗
PubMed1989

Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule (NCAM).

Monoclonal antibody (MAb) UJ13A, raised against 16-week-old human foetal brain, recognizes an antigen present on the majority of tissues of neuro-ectodermal origin. The binding profile of this antibody is similar to the known distribution of the neural cell adhesion molecule (NCAM). Although detailed immunohistological and immuno-cytochemical studies with the MAb have been published previously, we demonstrate here, through investigations of selected tissues and cell lines, that the binding profile of an anti-NCAM antiserum is similar to UJ13A. Additional indirect evidence suggesting that UJ13A recognizes NCAM comes from Northern blot analysis of cell lines either binding or not binding UJ13A as determined by indirect immunofluorescence. Only those cell lines known to bind UJ13A express high levels of NCAM mRNA. Western blot analysis of extracts of human brain show that UJ13A recognizes proteins of 180 and 140 kDa in addition to a very weak band of 120 kDa. This data corroborates the suggestion that UJ13A recognizes NCAM as these 3 isoforms of the protein are identified in human brain. Final confirmation of the specificity of UJ13A comes from the study of 3T3 fibroblasts transfected with a cDNA coding for the 125 kDa isoform of human muscle NCAM. UJ13A selectively recognizes these transfectants and binds to a 125 kDa protein isolated from the cells by Western blot analysis. Thus we conclude from these immunological, biochemical and molecular studies that the antigen recognized by the UJ13A MAb is the neural cell adhesion molecule.

Antibodies, Monoclonal↗
PubMed1989

Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma.

We describe reagents from 2 workshops which had been identified as recognizing the same or very similar antigens based on their tissue reactivity. Examination of their tissue specificity led us to the conclusion that this was similar to the expression of the neural cell adhesion molecule (NCAM). We also describe the use of a transfection-based assay to show that these reagents do recognize NCAM. 3T3 cells were transfected with a full-length clone of human NCAM. Indirect immunofluorescence studies showed binding of all related antibodies to the transfectants, but not to the control 3T3 cells. In addition, biochemical analysis using certain antibodies in the cluster confirm that they detect NCAM in the transfectants. Our study shows the benefits of using workshops to compare monoclonal antibodies and a molecular approach to define the antigens recognized by such reagents.

Antibodies, Monoclonal↗
PubMed1989

Distribution of dystrophin, nebulin and Ricinus communis I (RCA-I)-binding glycoprotein in tissues of normal and mdx mice.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene locus, appeared as an immunoreactive triplet of polypeptides in striated muscle tissues from normal mice on Western blot analysis. In smooth muscle tissues, an immunoreactive doublet of corresponding molecular weight was detected. No dystrophin was found in normal mouse brain, not even after enrichment for the Triton X-100 insoluble fraction. Dystrophin was absent from all corresponding tissues from the mdx mutant mouse strain which is known to lack dystrophin. The possibility that these immunoreactive bands represent isoforms is discussed. We have also investigated two other high molecular weight proteins which show secondary abnormalities in DMD muscle, namely nebulin and the 370 kDa Ricinus communis I lectin (RCA I)-binding glycoprotein. Nebulin levels were reduced in skeletal muscle from 6-week-old mdx mice but not in oesophagus from the same animals. By contrast, the RCA I-binding 370 kDa glycoprotein which is greatly reduced in DMD skeletal muscle was present in normal amounts in mdx skeletal muscle. These findings show, for the first time, that mdx myopathy differs from DMD myopathy not only morphologically, but also in its secondary biochemical abnormalities.

Animals↗
PubMed1989

Stability of protein pharmaceuticals.

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications. Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development. Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties. These properties pose difficult stability problems. A summary of both chemical and physical decomposition pathways for proteins is given. Chemical instability can include proteolysis, deamidation, oxidation, racemization, and beta-elimination. Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces. Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species.

Chemistry, Pharmaceutical↗
PubMed1989

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2.

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.

Adult↗
PubMed1989

Insulin and insulin-like growth factor I in brain tumors: binding and in vitro effects.

We have measured insulin and insulin-like growth factor I (IGF-I) binding in human gliomas, meningiomas, and normal brain and studied the effect of insulin on the morphology, proliferation, and differentiation of central nervous system tumor and normal fetal cells in culture. Specific 125I-insulin and 125I-IGF-I binding was demonstrated by competition-inhibition binding assays. Insulin binding was measured in plasma membrane preparations from 9 freshly isolated human meningiomas, 4 glioblastomas multiforme (GBMs), a low-grade glioma, a normal adult brain, and a fetal brain. IGF-I binding was measured in similar preparations from 5 meningiomas, 4 GBMs, a low-grade glioma, and a normal adult brain. Incubations were carried out at 4 degrees C for 18 to 20 hours. Meningiomas showed higher specific insulin binding per 0.25 mg of protein than GBMs (19% versus 3%, P less than 0.005), and this difference was not related to small differences observed in insulin degradation. By contrast, IGF-I binding was significantly higher in gliomas than in meningiomas (27% versus 12%, P less than 0.05). Also, IGF-I binding was significantly higher than insulin binding in GBMs (27% versus 3%, P less than 0.03); binding of both IGF and insulin was high in meningiomas. In normal adult brain IGF-I and insulin binding was 7 to 10%. The ability of insulin to support and enhance the growth of central nervous system tumor cells in culture was investigated. Cell cultures were derived from a freshly isolated glioblastoma, a low-grade glioma, and 3 meningiomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

[Membrane changes in Duchenne/Becker muscular dystrophy: lectin binding and localization of dystrophin].

An RCA I-lectin binding glycoprotein of Mr = 370 kD is missing from or altered in the plasma membrane of Duchenne muscular dystrophy (DMD) skeletal muscle. In the present study the carbohydrate chain of this glycoprotein was localized to the external face of the plasma membrane in human skeletal muscle, and dystrophin, the protein product of the DMD gene, was localized to the inner (cytoplasmic) face. On double labelled Western blots the two proteins appeared as closely apposed but distinctly separate bands. Comparison of the plasma membrane binding of five lectins with overlapping sugar specificities in skeletal muscle from patients with DMD and the allelic milder disease form, Becker muscular dystrophy (BMD) showed that the RCA I-binding glycoprotein also strongly binds to phytohaemagglutinin, thereby largely characterising the carbohydrate binding site. This glycoprotein was absent or altered in DMD and markedly reduced in clinically manifest BMD but present in preclinical clinical BMD. There was no general depletion of plasma membrane glycoproteins in DMD because consistent plasma membrane binding could be demonstrated by peanut and maclura pomifera lectin. The possible implications of these findings for the pathogenesis of DMD/BMD are discussed.

Adolescent↗