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Biomedical subjects

S A Khan

Publications and source records attributed to S A Khan.

At least 19 recordsLinked to original sources
PubMed1992

Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family.

The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.

Amino Acid Sequence↗
PubMed1992

Uncoupling of the DNA topoisomerase and replication activities of an initiator protein.

The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity of the initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.

Bacterial Proteins↗
PubMed1992

Type-specific antibodies to the platelet-derived growth factor receptors: role in elucidating the structural and functional characteristics of receptor types.

Two types of platelet-derived growth factor receptors have been cloned and sequenced. Both are glycoproteins with similar molecular weights. We have earlier established the ligand binding specificity, ligand-induced dimerization, and kinase activation of these two receptor types [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705; Kanakaraj et al. (1991) Biochemistry 30, 1761-1767]. In the present studies, we have investigated the biosynthesis, processing, and glycosylation of the alpha-receptor and compared its structural and functional characteristics to those of the beta-receptor. Unlike an anti-peptide antibody, AbP2 (amino acid residues 964-979), to the human beta-receptor which detects a phosphorylation-specific conformation of the receptor, an antibody, AbP alpha 2 (amino acid residues 956-971), to the corresponding region of the human alpha-receptor failed to do so. However, our studies revealed that the stability of the alpha-receptor is comparable to that of the beta-receptor. In addition, N-linked glycosylation of the alpha-receptor, like that of the beta-receptor, is not important in kinase activation. We have exploited the lack of an effect of N-linked oligosaccharides on the functioning of the alpha-receptor to develop a simple and rapid method for direct demonstration of ligand-induced noncovalently linked alpha-beta-receptor heterodimer formation. This method is based on the interaction between functionally active short and the long forms of two receptor types which can be resolved by denaturing gel electrophoresis.

Amino Acid Sequence↗
PubMed1992

Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway.

We have investigated the effects of steroidogenesis inducing protein (SIP) (Endocrinology (1990) 126, 3043-3052) on steroid production in MA-10 mouse Leydig tumor cells. Our results indicate that SIP results in the stimulation of progesterone production in MA-10 cells to the same extent obtained when maximal doses of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and dibutyryl cAMP (dbcAMP) are used. It was also observed that the increased progesterone production in response to SIP was not accompanied by an increase in intracellular cAMP levels as was seen following hCG stimulation. In addition, stimulation of progesterone production using maximal doses of LH, hCG and dbcAMP could be further increased by the addition of SIP to the incubation medium also indicating that this steroidogenic activity was acting through a differential signal transducing system than these hormones. That SIP was not acting through the cAMP second messenger pathway was also demonstrated by its lack of sensitivity to the neutralizing effects of a monoclonal antibody to LH as well as by its insensitivity to the protein kinase A inhibitor HA 1004 while both of these treatments significantly decreased LH and hCG stimulated steroid production. Lastly, SIP was unable to elicit the induction of several mitochondrial proteins which have previously been shown to be synthesized in MA-10 cells in response to LH, hCG and dbcAMP. Our results indicate that SIP stimulates the production of high levels of steroids through a signal transduction pathway which is distinct from that employed by trophic hormone stimulation in Leydig cells.

Animals↗
PubMed1992

Steroidogenesis-inducing protein interacts with transforming growth factor-beta to stimulate DNA synthesis in rat granulosa cells.

We have examined the effects of steroidogenesis-inducing protein (SIP), previously isolated from human follicular fluid, on the synthesis of DNA by granulosa cells isolated from diethylstilbestrol-primed immature rats. SIP alone had no effect but in conjunction with transforming growth factor-beta (TGF-beta) there was an increase in [3H]thymidine incorporation into granulosa cell DNA. The increase in [3H]thymidine into DNA was due to an increase in the number of labeled granulosa cells as assessed by autoradiography. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) interfered with the ability of SIP and TGF-beta to promote DNA synthesis. Previously, we reported that the growth-promoting action of follicle-stimulating hormone (FSH) on rat granulosa cells in vitro was dependent on TGF-beta, and EGF inhibited the actions of FSH plus TGF-beta on [3H]thymidine incorporation into DNA. Since the dependency of SIP on its interactions with TGF-beta and the ability of EGF to interfere with the process were similar to the properties reported for FSH, this raised the possibility that the actions of SIP were mediated through the accumulation of intracellular cAMP. However, when the hypothesis was tested, SIP had no effect on cAMP levels in the presence or absence of TGF-beta, under conditions in which FSH stimulated cAMP accumulation. In conclusion, DNA synthesis in rat granulosa cells is dependent on the presence of TGF-beta. In the presence of TGF-beta, FSH or SIP, acting through cAMP-dependent and cAMP-independent mechanisms respectively, can recruit more cells to enter the cell cycle and initiate DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.

Anilino Naphthalenesulfonates↗
PubMed1992

Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro.

The number of interstitial macrophages in the testis fluctuates according to age, increasing gradually during prepubertal development to reach 15-20% in the interstitial compartment in the adult rat. These macrophages are in close morphological association with Leydig cells. Macrophage products, interleukin-1 (IL-1) and tumor necrosis factor alpha stimulate and/or inhibit steroid production in cultures of Leydig cells. We have studied the effects of macrophage products on DNA synthesis in rat Leydig cells to investigate a possible paracrine role of testicular macrophage products on the proliferation of Leydig cells. Leydig cells isolated from 10-, 20-, and 70-day-old rats were cultured for 48 h in serum-free medium, washed, and treated with different cytokines for 18 h. The medium was then removed, fresh medium containing 0.5 microCi [3H]thymidine was added, and cells were incubated for 4 h prior to determining the incorporation of [3H]thymidine into DNA. Human recombinant IL-1 beta caused a dose-dependent stimulation in the incorporation of [3H]thymidine into DNA in the Leydig cells from 10- and 20-day-old rats but had no effect on DNA synthesis in interstitial cells from adult rats. Maximum stimulation of DNA synthesis in immature Leydig cells was observed with 1-2 ng/ml IL-1 beta. Autoradiography after incubation with [3H]thymidine showed a dramatic increase in the number of labeled Leydig cells after treatment with IL-1 beta (19.27 +/- 3.77% vs. 1.44 +/- 0.52% in control cultures) indicating that IL-1 beta recruited more cells to enter the cell cycle and initiate DNA synthesis. Human recombinant IL-1 alpha and tumor necrosis factor alpha also caused significant stimulation of DNA synthesis in Leydig cells but these cytokines were much less potent (1-10%) than IL-1 beta. IL-1 beta enhanced the effects of maximally effective concentrations of growth-promoting agents previously known to stimulate DNA synthesis in immature rat Leydig cells, i.e. human CG, steroidogenesis-inducing protein, and transforming growth factor alpha plus insulin. On the basis of these results it is concluded that IL-1 may play an important role in the proliferation of Leydig cells during prepubertal development in immature rats.

Animals↗
PubMed1992

The effects of acetazolamide on the ventilatory response to high altitude hypoxia.

Acetazolamide treatment ameliorates the symptoms of AMS; however, the mechanism by which this occurs is unclear. To examine the effects of acetazolamide on oxygenation, CO2 responsiveness and ventilatory pattern during acute exposure to HA, we studied two groups of subjects at SL and following rapid (less than 8 h) transport to HA. Acetazolamide or placebo tablets were given to groups 1 and 2, respectively, in a double-blind manner after baseline SL measurements; treatment was continued during HA exposure. There was no difference in the ventilatory pattern at HA, between the two groups. While the Ve achieved in response to CO2 at HA vs SL was much greater in each group the percent change from baseline at HA versus that at SL was not significantly different. The beneficial effects of acetazolamide in AMS are associated with a higher level of ventilation at HA and better oxygenation: CO2 chemosensitivity is not affected by acetazolamide at HA.

Acetazolamide↗
PubMed1992

Progestin and estrogen control of cathepsin D expression and processing in rat uterine luminal epithelium and stroma-myometrium.

Progestins increase the activity and rate of synthesis of cathepsin D, a lysosomal aspartyl protease, in the uterine luminal epithelium in ovariectomized rats. Western blot analysis of luminal epithelial proteins determined that the progestin, medroxyprogesterone acetate (MPA) increased the 43-kDa form of cathepsin D by 7-fold in 24 hr, whereas estradiol increased the amount of the same form by only 2-fold. To examine the precursor-product relationship between cathepsin D proteins in the luminal epithelium and stroma-myometrium after progestin or estradiol treatment, uterine proteins were prelabeled by incubation with [35S]methionine in vitro, cathepsin D was isolated by immunoprecipitation, and equal amounts of labeled cathepsin D were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After each hormonal treatment in each uterine tissue, a 48-kDa precursor was processed into a 44-kDa cathepsin D product. Endoglycosidase H digestion of [35S]methionine-labeled cathepsin D from the luminal epithelium and stroma-myometrium of medroxyprogesterone-treated rats shifted the molecular masses of the cathepsin D proteins by approximately 5.7 kDa. To examine the contribution of increased mRNA to increased rates of cathepsin D synthesis, we measured levels of cathepsin D mRNA in uterine tissues after progestin and estrogen treatment. Total RNA was isolated from the uterine luminal epithelium and from the stroma-myometrium. Northern blot analysis identified a single 2.2-kb RNA band corresponding to the size expected for cathepsin D mRNA. Medroxyprogesterone increased levels of cathepsin D mRNA in the luminal epithelium (greater than 17-fold) and in the stroma myometrium (3-fold), with maximum increases at 9 hr after treatment. Estradiol also increased cathepsin D mRNA levels in both uterine tissues, but by only 2-fold. No hormonal effects on liver cathepsin D mRNA were observed. Increases in cathepsin D synthesis and activity in uterine tissues in response to progestin and estrogen appear to depend in part upon increased levels of mRNA.

Animals↗
PubMed1992

Functional analysis of the N-terminal domain of Tat protein of the human immunodeficiency virus type 1.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of the viral long terminal repeat (LTR). The N-terminal region of Tat is rich in proline and acidic residues analogous to the activation domains of other transcription factors such as GAL-4 and CTF/NF-1. Several basic residues are also present in this region. To investigate the role of these structural features in the Tat-mediated trans-activation, we have chemically synthesized and evaluated Tat analogs with alanine or glutamine replacing one or more of these amino acid residues. Our data show that substitution of Glu-2, His-13, or all the proline in the Pro-Xaa3-Pro triad drastically reduced activity. In contrast, changes at Arg-7, Lys-12 and any one proline residue in the triad moderately reduced, and substitution of Lys-19 showed little effect on, activity. These results show that the native structure of the N-terminal 19 amino acid sequence is essential for Tat function, and that the overall topology of this domain and not the acidic residues alone appears necessary for trans-activation.

Amino Acid Sequence↗
PubMed1992

Impact on mortality of a community-based programme to control acute lower respiratory tract infections.

Acute lower respiratory tract infections (ALRIs) are a major cause of death among young children in developing countries. A targeted programme designed to treat children with ALRI was implemented in 1988 in a primary health care project in rural Bangladesh. In the 2 years preceding the introduction of the programme (1986-87), non-ALRI-specific health services were provided, including promotion of oral rehydration therapy, family planning, immunization of children and mothers, distribution of vitamin A, referral of severely sick children to field clinics, and nutritional rehabilitation of malnourished children. The targeted ALRI programme, which was in place in 1988-89, was based on systematic ALRI case detection and management by community health workers, who were linked to a referral system for medical support. These two levels of intervention have been evaluated by comparing the ALRI-specific mortality in the programme area and a neighbouring control area during the two periods. During the first phase (1986-87), the ALRI mortality among under-5-year-olds was 28% lower in the intervention than in the comparison area (P less than 0.01). During the second phase (1988-89), the ALRI mortality was 32% lower in the intervention area than during the preceding phase, while there was no significant difference for the comparison area. These findings suggest that in the study region the combination of specific and nonspecific interventions can reduce ALRI mortality by as much as 50% and the overall mortality among under-5-year-olds by as much as 30%.

Allied Health Personnel↗
PubMed1979

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Bacteriophage phi X 174↗