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S Deb

Publications and source records attributed to S Deb.

At least 109 records · Page 6Linked to original sources
PubMed1991

Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme.

In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.

Adrenal Glands↗
PubMed1991

A 269-amino-acid segment with a pseudo-leucine zipper and a helix-turn-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein.

The UL9 gene of herpes simplex virus (HSV) codes for a DNA-binding protein (OBP) that interacts sequence specifically with the origin of replication. This protein is essential for HSV DNA replication in cultured cells. The UL9 gene was cloned into a plasmid vector downstream of the SP6 RNA polymerase promoter. By using in vitro transcription and translation systems, a full-length OBP was synthesized. This synthetic protein is recognized by an antiserum generated against the C-terminal decapeptide of OBP and is functionally active in binding to OriS sequence specifically. The in vitro-synthesized protein has sequence specificity for binding similar to that found for the in vivo-generated OBP. A total of 14 in-frame deletion and insertion mutants of the UL9 gene were generated and expressed in vitro. Using these deletion mutants, we determined that the 269-amino-acid stretch defined by amino acids 564 to 832 localizes the OriS-specific DNA-binding domain. The N-terminal boundary is between amino acids 565 and 596, while the C terminus lies between amino acids 833 and 805. This segment contains a helix-turn-helix moiety and a pseudo-leucine zipper, neither of which alone can support DNA binding. The other leucine zipper from amino acids 150 to 173 is not required for the in vitro sequence-specific DNA-binding activity of OBP.

Adenosine Triphosphate↗
PubMed1991

Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.

Animals↗
PubMed1991

Psychopathology of people with mental handicap and epilepsy. I: Maladaptive behaviour.

One hundred and fifty mentally handicapped people (100 from hospital and 50 from the community) with epilepsy were studied along with an individually matched control group of 150 (100 from hospital and 50 from the community) non-epileptic mentally handicapped people. Behaviour was studied using the Profile of Abilities and Adjustment Schedule. Of the total population, 55.3% showed some type of severe behaviour problem. Although the epileptics showed slightly more severe behaviour problems than the non-epileptic group, there was no statistically significant difference between the two groups. Some differences emerged between the groups when subgroups of epileptics were studied.

Adult↗
PubMed1991

Psychopathology of people with mental handicap and epilepsy. II: Psychiatric illness.

The prevalence of psychiatric illness was studied in 150 epileptic mentally handicapped people (both hospital in-patients and living in the community) and a matched group of 150 non-epileptic controls. The Profile of Abilities and Adjustment (PAA) scale was used for the initial screening of psychiatric illness. Mildly to moderately handicapped individuals who had good communication skills and scored positively on the PAA schedule for psychiatric illness were interviewed using the PSE interview schedule. Severely mentally handicapped individuals who scored positively on the PAA's psychiatric illness subscale were observed and information was gathered from their medical notes and carers. A psychiatric diagnosis was made using DSM-III-R criteria. The non-epileptic group showed significantly more psychiatric illness than the epileptic group. Psychiatric illness was diagnosed in 25% of the cohort.

Adult↗
PubMed1991

Psychopathology of people with mental handicap and epilepsy. III: Personality disorder.

A group of 75 mildly to moderately mentally handicapped people with epilepsy, resident in both a hospital and the community, were studied together with an individually matched control group of non-epileptic patients. Their carers were interviewed to gather information for two observer-rated personality questionnaires, the Standardised Assessment of Personality (SAP) and the T-L Personality Behaviour Inventory. The two groups were compared with respect to the prevalence rates of various personalities. An abnormal personality score according to the SAP schedule was reported in 26% (n = 39) of the cohort, of which 28 (18.6% of the cohort) were personality disorders. A diagnosis of SAP abnormal personality was made in 46% of the in-patients and 6.5% of the community-based population. Of the cohort, 15% had an abnormal personality score according to the T-L schedule. No statistically significant difference emerged between the epileptic and the non-epileptic groups in the prevalence of either the SAP or T-L personality.

Adolescent↗
PubMed1991

Identification and immunochemical characterization of a major placental secretory protein related to the prolactin-growth hormone family, prolactin-like protein-C.

Trophoblast cells of the rat chorioallantoic placenta synthesize and secrete a number of proteins structurally related to pituitary PRL. During the purification of one member of the placental PRL family, PRL-like protein-A (PLP-A), we identified a major contaminating protein with a similar mol wt but possessing a more acidic pI (5.9-6.1) and different immunoreactivities. After isolation by two-dimensional gel electrophoresis, the more acidic contaminating protein was electroeluted, and its N-terminal amino acid sequence was determined by gas phase sequencing. The N-terminal sequence showed considerable homology with members of the PRL family, including characteristic positioning of cysteine residues at amino acids 4 and 11. The newly identified protein species have been termed PLP-C based on their structural similarity with pituitary PRL. The protein was further characterized by the generation of specific immunological probes. Antibodies were generated to electrophoretically purified protein and to a chemically synthesized peptide representing amino acids 11-32 of its N-terminal sequence. Each antiserum specifically recognized two major species migrating at approximately 25 and 29 kDa, respectively. The 29-kDa species specifically bound to Concanavalin-A, while the 25-kDa species failed to bind to the lectin. Furthermore, the 29-kDa species could be converted to the 25-kDa species by enzymatic deglycosylation. The antisera have also been used to examine the cell- and temporal-specific patterns of expression. The immunoreactive protein species (25 and 29 kDa) were localized primarily to spongiotrophoblast cells present in the junctional zone of the chorioallantoic placenta. Expression was initiated after midgestation and increased during the remaining part of gestation. In summary, PLP-C is a major secretory protein produced by spongiotrophoblast cells during the second half of gestation.

Animals↗
PubMed1990

Characterization of placental prolactin-like protein-A in intracellular and extracellular compartments.

Prolactin-like protein-A (PLP-A) was purified from medium conditioned by junctional zone explants dissected from the rat chorioallantoic placenta via concanavalin A affinity chromatography, sodium dodecyl sulfate-gel electrophoresis and two-dimensional gel electrophoresis. Monomeric PLP-A was found to be heterogeneous and consisted of two major molecular sizes, 29 and 33 kDa. The charge and size heterogeneity attributed to monomeric PLP-A could be accounted for by the addition of N-linked carbohydrate moieties to the PLP-A structure. Monomeric PLP-A lacked lactogenic bioactivities and several attempts to sequence 29 and 33 kDa PLP-A monomers proved unsuccessful. Biochemical characterization of PLP-A species present in various intracellular and extracellular compartments indicated that PLP-A is normally secreted as high molecular weight complexes and that PLP-A species were also targeted to the nucleus. PLP-A species isolated from placental cytosol or from serum of pregnant rats predominantly circulated as disulfide-linked high molecular weight complexes. PLP-A antipeptide antisera showed limited but specific reactivity with the high molecular weight PLP-A species. We have specifically identified a 29 kDa protein species in placental cell nuclei with three antibodies directed to three different regions of the predicted amino acid sequence of PLP-A. The 29 kDa nuclear immunoreactive protein had an equivalent electrophoretic mobility to the 29 kDa PLP-A protein of junctional zone cytosol. The 33 kDa PLP-A protein was not identified in the nucleus. The biological significance of the circulating high molecular weight PLP-A species or the nuclear PLP-A species remains to be determined.

Animals↗
PubMed1990

Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta.

The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a 1-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression.

Allantois↗
PubMed1990

Transplantable rat choriocarcinoma cells express placental lactogen: identification of placental lactogen-I immunoreactive protein and messenger ribonucleic acid.

The purpose of this investigation was to evaluate the abilities of a transplantable rat choriocarcinoma (Rcho) to produce placental PRLs. The Rcho tumor was analyzed biochemically and histologically for the expression of placental PRLs. Expression of placental PRL mRNAs was determined by Northern blot and in situ hybridization analyses. Expression of placental PRL proteins was determined by Western blot and immunocytochemical analyses. Histologically, Rcho tumors were characterized by the appearance of giant cell surrounding hemorrhagic regions. Female rats bearing the Rcho tumor beneath their kidney capsule showed extensive mammary gland development. The Rcho tumors expressed placental lactogen-I (PL-I) mRNA and protein, but there was no evidence of placental lactogen-II (PL-II), PRL-like protein-A (PLP-A), or PRL-like protein-B (PLP-B). Rcho PL-I mRNA and proteins migrated as a 1-kilobase species and a 36- to 40-kDa species similar to those expressed by normal rat trophoblast tissues. The cell type responsible for Rcho PL-I production was the giant cell, similar to that observed in normal rat trophoblast tissues. In summary, we have demonstrated the production of PL-I by a transplantable rat choriocarcinoma (Rcho). The Rcho tumor resembles rat trophoblast tissue at early postimplantation stages (days 6-10 of gestation) and may be a useful tool for studying placental PRL expression during trophoblast differentiation.

Animals↗
PubMed1990

Critical self-association of bile lipids studied by infrared spectroscopy and viscometry.

Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy and viscometry were applied to study the micellization of two bile lipids, sodium taurochenodeoxycholate (NaTCDC) and sodium glycocholate (NaGC), in aqueous solutions. The CH2 stretching bands of the bile lipid hydrocarbon region were shifted to higher frequencies suggesting initial critical micellization at 2.5 mM for NaTCDC and 9 mM for NaGC. An abrupt enhancement of the absorption intensity of the CH3 groups of the sterol rings in bile lipids were under conformational strain at 3.5 mM NaTCDC and 9 mM NaGC. Viscometry measurements showed abrupt changes in viscosities in the region of critical micellar concentration (CMC) of both bile lipids. Both infrared and viscometry studies confirmed the onset of conformational strains in tightly packed lipid micelles at their CMC. In addition, FTIR/ATR spectroscopy has defined the specific hydrophobic interactions which bring about critical micellization of bile lipids.

Bile↗
PubMed1989

Placental prolactin-like protein A. Identification and characterization of two major glycoprotein species with antipeptide antibodies.

The purpose of this investigation was to develop immunologic probes to prolactin-like protein A (PLP-A) that could be used to characterize the protein and its distribution in various tissues. Five oligopeptides corresponding to different regions of the predicted PLP-A amino acid sequence (peptides 1-13, 62-76, 101-114, 129-145, and 152-164) were chemically synthesized by solid phase methodology. The peptides were purified to homogeneity by reverse phase high pressure liquid chromatography and coupled to keyhole limpet hemocyanin. The peptide-keyhole limpet hemocyanin conjugates were used to immunize rabbits. Immune responses were monitored by enzyme-linked immunoassay. Reactivity of the antipeptide antisera with placental proteins was determined by immunoblotting and immunoprecipitation analyses. All of the peptides except peptide 1-13 yielded significant immune responses. Antisera to peptides 101-114, 129-145, and 152-164 each specifically recognized proteins of Mr 29,000 and 33,000 from cytosol preparations of rat placental tissue and showed limited or no cross-reactivity with other members of the prolactin-growth hormone family. Three experiments were performed to determine whether the Mr 29,000 and 33,000 species were glycosylated derivatives of an Mr 25,000 precursor. Treatment of placental cytosolic preparations with N-Glycanase prior to immunoblotting resulted in the identification of only an Mr 25,000 species. It was also determined that the Mr 29,000 and 33,000 species specifically bound to concanavalin A. Furthermore, tunicamycin shifted the synthesis of PLP-A by placental explants from the Mr 29,000 and 33,000 forms to the Mr 25,000 species. The Mr 29,000 and 33,000 species were identified in serum obtained from pregnant and fetal rats but not in serum from nonpregnant females or males. We conclude that PLP-A is expressed in rat placenta. An Mr 25,000 precursor (predicted from PLP-A cDNA and these results) is glycosylated to either the Mr 29,000 or 33,000 form, both of which predominate in placenta and in circulation.

Amino Acid Sequence↗
PubMed1989

Analysis of Ori-S sequence of HSV-1: identification of one functional DNA binding domain.

Using gel retardation assays, we have detected an Ori-S binding activity in the nuclear extract of HSV-1 infected Vero cells. The sequence-specific DNA binding activity seems to be identical to that described by Elias et al. (Proc. Natl. Acad. Sci. USA 83: 6322-6326, 1986). This activity fails to retard a mutant origin DNA that has a 5 bp deletion in the reported protein binding site along with an A to T substitution at a position 16 base-pairs away from the site. This mutant also failed to replicate in a transient replication assay, thus correlating binding of the factor on the origin to replication efficiency. Using crude nuclear extracts as the source of the factor and with the help of footprint and gel retardation analyses, we confirmed that protection is only observed on the preferred site of binding on and near the left arm of the Ori-S palindrome. In order to analyze the sequence specificity of the binding we have generated a set of binding site mutants. Competition experiments with these mutant origins indicate that the sequence 5'-TTCGCACTT-3' is crucial for binding.

Animals↗
PubMed1989

Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II.

The purpose of this investigation was to develop specific immunologic probes to rat placental lactogen-II (PL-II) and to use the immunologic probes to further characterize rat PL-II. Five oligopeptides corresponding to different regions of rat PL-II (amino acids 1-13, 56-70, 89-103, 107-118, 150-164) were chemically synthesized by solid phase methods and purified to homogeneity by reverse phase high performance liquid chromatography. The synthetic peptides were coupled to keyhole limpet hemocyanin (KLH) and the peptide-KLH conjugates were used to immunize rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA), electrophoresis and immunoblotting analyses. Each of the antipeptide antisera showed reactivity with the entire rat PL-II protein; however, the extent of the reactivities of each antiserum with rat PL-II was dependent on the conformational state of rat PL-H. Antisera directed to amino acids 56-70 showed the best reactivity toward each of the conformational states of rat PL-II tested. Antibodies generated to the entire rat PL-II protein specifically recognized the 56-70 amino acid sequence but showed limited reactivity with synthetic peptide corresponding to amino acids 1-13, 89-103, 107-118, and 150-164 of rat PL-II. Antisera to amino acids 56-70 of rat PL-II were specific for PLs as demonstrated by their recognition of rat PL-II, mouse PL-II and human PL and by their lack of reactivity with rat pituitary prolactin and growth hormone and with a series of other synthetic peptides to rat PL-II and rat prolactin-like protein-A. The immunorecognition of human PL was restricted to antipeptide antibodies directed to amino acids 56-70 of rat PL-II. The chemically synthesized peptides representing various regions of rat PL-II did not show significant interactions with prolactin receptors, and antisera directed to the peptides failed to interfere with the binding of either rat PL-II or human PL to prolactin receptors. In summary, we have generated a series of immunologic probes for studying the structure of rat PL-II. The sequence comprising amino acids 56-70 of rat PL-II was shown to make up at least part of an epitope for rat PL-II and to be a region of significant structural homology with mouse PL-II and human PL.

Amino Acid Sequence↗
PubMed1989

Preferential binding of simian virus 40 T-antigen dimers to origin region I.

The sequence components that direct high-affinity binding of simian virus 40 (SV40) T antigen to SV40 origin region I are composed of two recognition pentanucleotides separated by a spacer. This region has binding sites for two T-antigen monomeric units. We extended the tripartite region I sequence by one and two sets of spacers and pentanucleotides and also shortened the region by one pentanucleotide. Our T-antigen-binding studies with these constructs show that the protein has a strong preference for binding to an even rather than an odd number of pentanucleotides separated by spacer sequences. Gel retardation assays reveal that the size of the complex formed between the 17-base-pair region I sequence and T antigen did not increase when the sequence was extended with one spacer-pentanucleotide sequence but did increase with two such units. DNase I footprinting and fragment assay experiments indicate that the protein did not protect a pentanucleotide that was not paired with another pentanucleotide. The unpaired pentanucleotide resumed its binding activity when it was paired with a spacer and another pentanucleotide sequence. We propose that T antigen binds to region I as a preformed dimer.

Animals↗
PubMed1989

Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta.

The rat chorioallantoic placenta is comprised of two morphologically distinct regions: the junctional zone and the labyrinth zone. The purpose of this investigation was to determine the relative contributions of trophoblast cells in each of these regions to the expression of placental lactogen-II (PL-II) and PRL-like protein-A (PLP-A) during development, mRNA expression was estimated by Northern blot analysis, whereas, protein expression was estimated by electrophoresis, immunoblotting, and immunocytochemical analyses. The immunochemical analyses used antipeptide antisera directed to amino acids 56-70 of PL-II and amino acids 152-164 of PLP-A. Northern and immunoblotting analyses indicated that both PL-II mRNA and protein expression were maximal in the junctional zone on day 13 of gestation and declined as gestation proceeded. In contrast, PL-II mRNA and protein expression in the labyrinth zone were low on day 13 and increased as gestation advanced. PL-II was specifically localized to giant cells. At midgestation, PL-II-positive giant cells were identified bordering the uterine decidua in the junctional zone and choriovitelline placenta. As gestation advanced. PL-II-positive cells were also localized to the labyrinth zone. Immunoreactivity was restricted to the cytoplasm of PL-II-positive cells. PLP-A mRNA and protein were predominantly expressed in the junctional zone of the chorioallantoic placenta. Expression of PLP-A increased as gestation advanced. PLP-A was specifically localized to giant and spongiotrophoblast cells of the junctional zone. Immunoreactivity was found in both cytoplasmic and nuclear compartments of PLP-A-positive cells. In summary, PL-II expression shifts from the junctional to the labyrinth zone during pregnancy, whereas PLP-A is predominantly expressed in the junctional zone during the latter third of pregnancy. Both hormones are produced by giant cells of the junctional zone, but only PL-II is expressed by choriovitelline and labyrinthine trophoblast cells. PLP-A is also expressed by spongiotrophoblast cells of the junctional zone. These findings provide insights into the process of placental morphogenesis.

Allantois↗