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P Banerjee

Publications and source records attributed to P Banerjee.

At least 19 recordsLinked to original sources
PubMed1992

Enrichment of saturated fatty acid containing phospholipids in sheep brain serotonin receptor preparations: use of microwave irradiation for rapid transesterification of phospholipids.

During enrichment of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-binding serotonin 5-HT1A receptors from sheep brain gray matter (membrane isolation, detergent solubilization and reconstitution into vesicles) a consistent and striking increase in the composition of saturated fatty acids was observed in phospholipids which were coisolated with the receptors. A rapid procedure has been developed for the methylation of free and phospholipid linked fatty acids which were thus analyzed by gas chromatography-mass spectrometry (GC/MS). Esterification of free fatty acids and transesterification of phospholipid linked fatty acids were achieved with 14% boron trifluoride in methanol (BF3-CH3OH) in 20 s and 50 s, respectively, under low power microwave irradiation (60 W) with a post-reaction cooling of less than 5 min. This is in contrast to the conventional method of heating in a boiling water bath for 10-15 min with BF3-CH3OH which is inevitably preceded by time-consuming and inconvenient clamping of vials and followed by cooling for 10 min before the vials can be safely opened. Analysis of fatty acid profiles in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) from egg yolk, phosphatidylinositol (PI) from bovine liver and phosphatidylserine (PS) from bovine brain by both techniques showed comparable results. During detergent solubilization of sheep brain gray matter, the overall proportion of saturated fatty acids in PE (major lipid), PI, PC (major lipid) and PS increased from 50-60% in sheep brain phospholipids to 70-75% in 1.5% CHAPS solubilized, reconstituted and biologically active serotonin 5-HT1A preparations. In sharp contrast, the proportions of saturated fatty acids in 1.5% Triton X-100 solubilized PE (48.1%) (major lipid), PI (63.6%), PC (60.6%) (major lipid) and PS (62.2%) were not significantly different from those in the original sheep brain membranes. Strikingly, this was coupled with the occurrence of very low levels of 5-HT1A receptor activity in the Triton X-100 solubilized preparations. The abundance of 5-HT1A sites in the enriched vesicles obtained only from the CHAPS-solubilized preparations was further confirmed by specific radiolabeling of a 58-kDa polypeptide by the 5-HT1A specific ligand p-aminophenylethyl-m-trifluoromethylphenylpiparazine (PAPP) which was coupled to a 125I-labeled, photoreactive, heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropiona te (SASD). Thus CHAPS-solubilized 5-HT1A receptor preparations are depleted in the more rigid lipids such as sphingolipids and cholesterol, (Banerjee et al. (1990) Biochim. Biophys. Acta 1044, 305-314), but are enriched in vesicle-stabilizing, phospholipid-linked saturated fatty acids which in turn probably stabilize the heptahelical, membrane bound 5-HT1A receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Raf-1 activates MAP kinase-kinase.

The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.

3T3 Cells↗
PubMed1992

Evidence for lipase abnormality: high levels of free and triacylglycerol forms of unsaturated fatty acids in neuronal ceroid-lipofuscinosis tissue.

Total lipid obtained from normal and different forms of neuronal ceroid-lipofuscinoses (NCL) tissues was analyzed by high performance thin layer chromatography (HPTLC). We observed a large (greater than 6-fold) increase in a lipid band corresponding to triolein for NCL dog pancreas and spleen and juvenile human NCL brain and infantile NCL spleen. The accumulation was less pronounced for the brain samples but apart from increased dolichol-monophosphate levels, other lipids appeared normal. Normal dog, goat, or human spleen contained virtually no triacylglycerol, and of the pathological controls, beta-mannosidosis goat spleen showed no triacylglycerol band at all. A sample of human spleen from a patient with lymphoma-associated splenomegaly displayed a strong triacylglycerol band, but gas chromatography-mass spectrometry (GC/MS) of the bands showed an equal increase in both saturated and unsaturated fatty acid containing triacylglycerols in the splenomegaly sample, in keeping with the notion of non-specific fat deposition in damaged tissue. In contrast, in all the NCL samples (spleen, pancreas, and brain) a prominent increase in the proportion of unsaturated fatty acids was observed in both free fatty acid and/or triacylglycerol bands following GC/MS. The NCL-English setter dog pancreas showed a major presence of oleic acid (18:1) (twofold increase) as compared to normal, while dog and infantile human NCL spleen samples and juvenile Batten brain (human) displayed a robust increase in linoleic acid (18:2) and sometimes in oleic acid and arachidonic acid (20:4) (for infantile human NCL spleen). For the infantile human NCL spleen sample an increase in linoleic acid in both free fatty acid (3.2-fold) and triacylglycerol (10-fold) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Molecular basis of an adult form of beta-hexosaminidase B deficiency with motor neuron disease.

A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta-Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons.

Adult↗
PubMed1991

Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

Amino Acid Sequence↗
PubMed1991

Medulloblastoma: long-term follow-up of patients treated with electron irradiation of the spinal field.

Thirty-two patients with posterior fossa medulloblastoma underwent treatment with electron irradiation to the spinal field. The 5- and 10-year actuarial survival rates were 57% and 50%, respectively. Late complications observed in the 15 patients followed up for more than 5 years were short stature (six patients), decreased sitting-standing height ratio (four patients), scoliosis (two patients), poor school performance (seven patients), xerostomia (one patient), esophageal stricture (one patient), pituitary dysfunction (four patients), primary hypothyroidism (one patient), bilateral eighth-nerve deafness (one patient), and carcinoma of the thyroid (one patient). Complications following treatment with electrons to a spinal field are compared with reported complications following treatment with photons to the spinal field. Although short-term reactions were minimal, the authors found no difference in late complications. More sophisticated treatment planning may show such a long-term benefit in the future.

Adolescent↗
PubMed1990

Asymmetric extraction of membrane lipids by CHAPS.

We have characterized and quantitated the lipids which are cosolubilized with serotonin 5-HT1A sites from sheep brain using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Dialysis of the CHAPS extract produced a [3H]8-hydroxy(2-di-n-propylamino)tetralin [( 3H]8-OH-DPAT) binding vesicular preparation of the protein. Quantitative analysis of the lipids present in the CHAPS extract by HPTLC and transmittance-densitometry revealed extraction of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidyl serine (PS) and phosphatidic acid (PA) in striking preference over cholesterol, galactosylceramides, sulfatides and sphingomyelin. All lipids present in the clear CHAPS-extract were coeluted with the [3H]8-OH-DPAT binding preparation were separated by centrifugation, 95-100% of the [3H]8-OH-DPAT binding protein was retained in the vesicle-containing pellet. The supernatant contained small amounts of cholesterol, PE and PC, but virtually no PS, PI, or PA, whereas the vesicular pellet contained all the lipids mentioned, indicating that PS, PI and PA are more tightly bound to the vesicles than PE, PC and cholesterol. SDS-PAGE analysis of the pellet revealed two major protein bands, at 58 kDa and 33.5 kDa, respectively. Our report outlines a simple and improved densitometric assay used for the first detailed analysis of lipids cosolubilized with an active, membrane protein, and also, a simple assay for CHAPS.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1990

Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase.

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.

Amino Acid Sequence↗
PubMed1989

Critical evaluation of conventional abdominal closure with single-layer closure in adult and elderly.

Two different techniques of abdominal closure, conventional and single-layer, were studied on comparative basis in 55 cases each. Single-layer closure was found to have definite advantage over conventional closure as regards operating or healing time, feasibility, ease and postoperative morbidity. Single-layer closure took 8-10 minutes in comparison to 18-20 minutes required by conventional method. Incidence of burst abdomen was found to be 3.6% in the former and 7.27% in the latter. In only one case of conventional closure incisional hernia occurred whereas none occurred in case of single-layer closure. Moreover complications of scar and delayed healing were less in single-layer closure.

Abdomen↗
PubMed1976

Ewing's sarcoma.

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Adolescent↗