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Biomedical subjects

S Habib

Publications and source records attributed to S Habib.

At least 37 records · Page 2Linked to original sources
PubMed1998

The host factor polyhedrin promoter binding protein (PPBP) is involved in transcription from the baculovirus polyhedrin gene promoter.

Hypertranscription and temporal expression from the Autographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an alpha-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750-2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405-4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda (Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polh promoter. Replacement of PPBP cognate sequences within the polh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the polh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in trans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.

Animals↗
PubMed1997

Involvement of host factors in transcription from baculovirus very late promoters -- a review.

The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin. This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes. Regulation of transcription from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified. Fresh insight into host-virus interactions during baculovirus pathogenesis is now offered by the identification of insect host factors that interact with transcriptionally essential motifs of these promoters as well as cis-acting enhancer-like elements upstream from the promoter.

Base Sequence↗
PubMed1997

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system.

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP3), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca2+ channel, the IP3 receptor (IP3R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf (S. frugiperda) cell system that can be used to look at IP3R function. Agonist-evoked changes in intracellular Ca2+ levels [Ca2+]i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vm1AchR). Furthermore, we have constructed a recombinant BV (vIP3R), with the core of the IP3R ligand-binding domain from the Drosophila IP3R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vm1AchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP3R construct and vm1AchR have been used to assay the modulation of IP3R-mediated Ca2+ release, by the ligand sink.

Animals↗
PubMed1996

A 38-kDa host factor interacts with functionally important motifs within the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region (hr1) DNA sequence.

We recently demonstrated that the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region (hr1) enhances transcription from the viral polyhedrin promoter and also functions as a putative origin of replication (ori). Hr1, carrying five 28-base pair core palindrome units, has also been mapped with respect to its enhancer and ori functions (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737-747). A 38-kDa host factor termed hr1-binding protein (hr1-BP) binds with high specificity and affinity (Kd approximately 6.5 x 10(-11) M) to functionally important motifs within hr1. The core palindrome as well as sequences immediately flanking it are required for this interaction. Divalent cations are not essential, and ionic interactions play only a minor role in complex formation. hr1-BP binds through the minor groove of the double helix to multiple sites within hr1, and binding occurs as a function of the number of modules within hr1. Phosphorylation of hr1-BP is important for host factor-hr1 interaction. Hr1-BP differs in several respects from the other host factor, polyhedrin promoter-binding protein, described previously (Burma, S., Mukherjee, B., Jain, A., Habib, S., and Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750-2757). When hr1-BP was sequestered out, in vivo, by a plasmid carrying hr1 alone, the hr1-mediated enhancement of reporter expression was abolished, demonstrating that the binding of hr1-BP may be crucial for the enhancer activity of the dual function hr1 element.

Base Sequence↗
PubMed1996

Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response.

This paper reports the expression of a previously described gene [Nath and Laal, Nucleic Acids Res. 18 (1990) 4935], currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system. The produced protein moved as a 95-kDa band on SDS-PAGE. An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence. A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon. The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding. Of functional significance was its immunoreactivity in human subjects with mycobacterial infection. Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

Adenosine Triphosphate↗
PubMed1996

Bifunctionality of the AcMNPV homologous region sequence (hr1): enhancer and ori functions have different sequence requirements.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) homologous region sequence hr1 is a putative origin of replication (ori) sequence and can also function as a transcriptional enhancer for delayed-early genes. We demonstrate that this 750-bp sequence, carrying five 28-bp core palin-dromes, enhances expression from the very late polyhedrin promoter up to 11-fold in a classical enhancer fashion in transient expression assays. Enhancement is at the level of transcription, as evident from RNase protection assay analysis. It is mediated by an alpha-amanitin-insensitive RNA polymerase from the authentic polyhedrin promoter transcription start site and follows the temporal activation profile characteristic of the polyhedrin promoter. Three lines of evidence conclusively demonstrated that hr1 acts typically as an enhancer of polyhedrin gene transcription independent of its role as an ori: (i) linearized hr1-reporter plasmids, incapable of replicating in the host cell, could enhance transcription from the promoter; (ii) reporter plasmid copy number was not affected by the presence of aphidicolin during transfection; (iii) reporter plasmid DNA recovered from Sf9 cells was sensitive to Dpn I confirming its unreplicated state in the transfection regime followed by us. Molecular dissection of the hr1 sequence elements revealed that a core palindrome alone can function as an ori sequence whereas a palindrome along with flanking sequences is essential for the enhancer activity. Enhancement of luciferase expression from the polyhedrin promoter is a function of the number of core palindromes and flanking sequences. Our results demonstrate that hr1, which has several motifs for enhancer binding proteins and transcription factors, has a dual role associated with both DNA replication and transcriptional enhancement.

Animals↗
PubMed1995

Perception and social consequences of tuberculosis: a focus group study of tuberculosis patients in Sialkot, Pakistan.

Treatment defaulting is one of the major causes of the failure of TB control programs. In Bethania Hospital, Sialkot, defaulting rates are high: 72% for the standard 12 months course and 56% for the 8 months course. Attrition is especially important in the first weeks of treatment: < 70% of the patients start the 10th week of treatment. A focus group discussion study has been carried out to gain a better understanding of the impact of social stigmatization, treatment cost and pregnancy on defaulting. The study population consisted of 3 male and 3 female groups each with 8 hospitalized TB patients. The study shows that TB is perceived as a very dangerous, infectious and incurable disease. This perception has many social consequences: stigmatization and social isolation of TB patients and their families: diminished marriage prospects for young TB patients, and even for their family members: TB in one of the partners may lead to divorce. Due to fear patients often deny the diagnosis and reject the treatment. While both male and female TB patients face many social and economical problems, female patients are more affected. Divorce and broken engagements seem to occur more often in female patients. Females are usually economically dependent on their husbands and family in law, and need their cooperation to avail of treatment. The belief that pregnancy enhances the risk for relapse decreases their marriage prospects. Pregnancy is also a reason for stopping TB treatment as both are considered as incompatible. The findings of this study reveal the urgent need for a health education campaign to convince the general population that tuberculosis is curable. All health care providers should act as destigmatizers.

Adolescent↗
PubMed1994

An unusual 30-kDa protein binding to the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus.

Gel retardation assays using nuclear extracts from the Autographa californica nuclear polyhedrosis virus-infected Sf21 (Spodoptera frugiperda) insect cell line revealed a host factor (polyhedrin promoter-binding protein or PPBP) that binds to the polyhedrin gene promoter. A hexanucleotide sequence (AATAAA) within the promoter is important for binding in association with neighboring elements, some of which are contributed by the sequence TAAGTATT present at the transcription start point. PPBP was affinity purified and appears to be an unusual DNA-binding protein with respect to its stability (binding was obtained at NaCl concentrations and temperatures ranging from 0.2 to 2 M and 0 to 65 degrees C, respectively), high binding affinity (binding even in the absence of nonspecific DNA) with an apparent dissociation constant of approximately 3.7 x 10(-12) M, and high specificity (binding was unaffected in the presence of a 50,000 times excess of nonspecific DNA). From UV cross-linking and Southwestern analyses the molecular mass of PPBP was estimated to be approximately 30 kDa. PPBP is phosphorylated and may have a regulatory function because dephosphorylation abolished DNA binding activity. Differences in PPBP characteristics between nuclear extracts from Sf21 and from Bm5 (Bombyx mori) cell line suggested that additional factors may be involved in the interaction of PPBP with the polyhedrin promoter.

Animals↗