Molecular characterization of senescence marker protein-30 gene promoter: identification of repressor elements and functional nuclear factor binding sites.

BACKGROUND: Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene. RESULTS: We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. Twenty eight transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5' and 3' -deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/+157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/-368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/-148 bp) and a C/EBPbeta (-190/-177 bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity. CONCLUSION: These studies provide novel insights into the mechanism that regulate SMP30 gene expression.

Cloning, expression, and functional analysis of rat liver cytosolic inorganic pyrophosphatase gene and characterization of its functional promoter.

Inorganic pyrophosphate (PPi) is formed in several metabolic processes and its hydrolysis by the ubiquitously expressed enzyme inorganic pyrophosphatase (iPPase) is essential for the reactions to proceed in the direction of biosynthesis. Recently, we have reported differential expression and activity of cytosolic iPPase in rat liver with aging. In this article we report the cloning of the coding region of rat liver cytosolic iPPase gene in a bacterial expression vector, its expression, purification, and functional analysis by in-gel enzyme assay. SDS-PAGE and Western blot analysis of this expressed protein revealed that its molecular weight (MW) is approximately 33 kDa, while in-gel assay showed that it is functionally active just as the liver cytosolic iPPase. We have determined the genomic organization of this gene by genome blast approach. We have also cloned and characterized its proximal approximate 1 kb functional promoter (-1009 to +82) by transient transfection and luciferase assay of different 5'-deleted iPPase promoter-luciferase constructs and also established its transcription start site by primer extension analysis, along with protein-DNA interaction studies for a few putative transcription factor binding sites.

Polymorphism study of rhoptry associated membrane antigen (RAMA) gene of Plasmodium falciparum--a putative vaccine candidate.

The antigenic diversity of Plasmodium falciparum is one of the major obstacles to antimalarial vaccine development. Thus, it becomes obvious to search for a protein that is fairly conserved and is necessary for the parasite to survive in the host cell. Rhoptry associated membrane antigen (RAMA) plays a potential role in parasite biology and invasion. The C-terminal end of RAMA is shown to have protective immune responses and binds to the erythrocyte membrane. In this work, we have studied the polymorphism of RAMA gene in the C-terminal end from 1525 to 3196 nucleotide (nt) in 230 samples. The presence of few variants suggests RAMA to be under a balancing selection. The above criterion of restricted antigenic diversity and a protective immune response towards the C-terminal end makes RAMA a strong vaccine candidate.

The development and evaluation of a single step multiplex PCR method for simultaneous detection of Brugia malayi and Wuchereria bancrofti.

A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites, Brugia malayi and Wuchereria bancrofti, from blood samples and mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188bp (BM) and 129bp (WB) DNA fragments, specific to B. malayi and W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both B. malayi and W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.

Altered expression of trefoil factor 3 and cathepsin L gene in rat kidney during aging.

Alterations in a wide array of physiological functions are normal consequences of aging. It is likely that, decline in cellular and physiological functions that occur during aging are the net result of age related differential gene expression and their consequent down stream effects. In this report we demonstrate that in aged kidney there is a decrease in the expression of trefoil factor 3 gene and an age-related increase in the expression of cathepsin L gene as revealed by differential display PCR (DD-PCR) and northern blot analysis. Trefoil factor 3 is mainly expressed in the alimentary canal and protects it from the degradative effect of HCl by stimulating the goblet cells to synthesize mucin. Though the exact role of trefoil factor 3 in kidney is not known, we speculate that it has a protective role in kidney. Cathepsin L is a cysteine protease which degrades connective tissue proteins like collagen, elastin and fibronectin. Increase in the expression of cathepsin L in aged kidney leading to considerable loss of organ function in old age. Down regulation of trefoil factor 3 and up regulation of cathepsin L may contribute to lack of protection and increased age related tissue damage to kidney in aging.

An efficient PCR--SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum.

The spread of chloroquine resistance throughout the world poses a major problem in combating malaria. In the present study, an efficient polymerase chain reaction-single strand conformational polymorphism (PCR--SSCP)-based assay detected the PfCRT K76T point mutation, which is a marker for chloroquine resistance. For the first time, we have used a PCR--SSCP-based technique to identify the mutation in a single-step labelling reaction during PCR and SSCP gel electrophoresis. This assay is 100% efficient, giving no false-positive or -negative results, and can be carried out within a short bench time. We have successfully analysed 120 natural isolates using the PCR-SSCP method for detection of the chloroquine resistance marker and found 91 of the 120 samples to show the PfCRT T76 mutation, and 71% (65 of the 91 samples) showed a positive correlation with chloroquine resistance from the clinical data of the patients. The PCR-SSCP technique can also be applied for the detection of new haplotypes of the PfCRT gene and surveillance of chloroquine-resistant malaria in malaria-endemic localities around the world.

Expression of Stat5A in tobacco chewing-mediated oral squamous cell carcinoma.

Oral squamous cell carcinoma (oscc) is the fifth most common cancer worldwide and the number of cases is increasing regularly in the developing world. The effective detection of oscc at its early stages becomes necessary for proper treatment due to limited understanding of the critical pathways during oncogenesis. Signal transducer and activators of transcription (Stats) are an important group of transcription factors, which contribute to tumorigenesis due to their intimate connection to growth factor signalling, apoptosis, and angiogenesis. They also play a critical role in immune responses and hence defective Stat signalling could favour tumour development by compromising immune surveillance. The role of Stat5A in mammary gland carcinoma and leukaemia has already been reported. We for the first time report here the constitutive activation of Stat5A as one of the early events in tobacco mediated-oscc in the eastern Indian population, which can be used as a potent prognostic molecular marker.

Increased phosphorylation and decreased level of IkappaBalpha during aging in rat liver.

Nuclear factor kappa B (NFkappaB) is an evolutionary conserved transcription factor, which coordinates various metabolic processes triggered by innate and adaptive immune responses. Supakar et al. (J. Biol. Chem. 270: 837-842, 1995) had reported a 10-fold increase in DNA binding activity of NFkappaB in liver of old rats. In this study, we have analyzed the changes in the level of NFkappaB, inhibitor of NFkappaB (IkappaBalpha), phosphorylated-IkappaBalpha (p-IkappaBalpha) and IkappaB kinase (IKK) in rat liver during aging by reverse transcription polymerase chain reaction and/or western blotting. Here we demonstrate that there is an age-dependent increase in the level of p-IkappaBalpha with concomitant decrease in the level of IkappaBalpha, which may be correlated with increased inflammation, oxidative stress and higher level of activated NFkappaB in rat liver in old age.

Genetic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum in different malaria-endemic localities.

A number of stage-specific antigens have been characterized for vaccine development in Plasmodium falciparum malaria. The polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a major asexual blood stage malaria vaccine candidate antigen. In the present study, we analyzed the impact of hyperendemic malaria transmission, mesoendemic malaria transmission, and multiple infection on allelic diversity. We have used a simple strategy of polymerase chain reaction amplification and slot-blot hybridization to analyze variable regions of block-2, block-4 and blocks 6-10 of the MSP-1 gene. The allelic types of isolates collected from regions of hyperendemic malaria transmission (RHEMT) and mesoendemic malaria transmission (RMEMT) were compared. In RHEMT, 20 of 24 possible gene types were found among 163 isolates and more than one allelic type was found in 82 (50.3%) of the isolates. Thirteen of 24 possible gene types were found among 125 isolates in RMEMT and 27 (21.6%) of them contained more than one allele type. Our results suggest for the first time that the allelic distribution or allelic diversity and chances of finding multi-strain parasites in isolates in an area vary with the rate of transmission. Analyses of isolates containing more than one strain of parasite suggest that allelic types are randomly distributed, no specific type of alleles predominately show multi-strain infection, and neither strain of the parasite affect the process of infection and development of another.

Decreased expression of the pancreatic secretory trypsin inhibitor II gene during aging of the rat liver.

The genetic constitution and differential gene expression of an organism play important roles in controlling the species-specific rate of aging and the maximum life span potential. We utilized a differential-display polymerase chain reaction technique to identify the age-dependent expression of genes in the rat liver. We demonstrate in this report, for the first time, that expression of the pancreatic secretory trypsin inhibitor II (PSTI-II) gene declines drastically during aging. We confirmed this decrease by Northern blot analysis. Low PSTI-II levels in aged animals might result in a lack of protection from prematurely activated trypsin-like proteases, which would thus enhance inflammation.

Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates.

Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.

Identification of telomerase activity in gametocytes of Plasmodium falciparum.

Telomerase, a specialized cellular reverse transcriptase, compensates the chromosome shortening during the replication of most eukaryotic cells and contributes to cellular immortalization in cell culture (in vitro) and cancerous cell (in vivo). In the present study, the telomerase activity in the gametocytes of Plasmodium falciparum was investigated. Here, we report for the first time, the presence of telomerase activity in the gametocytes of P. falciparum using P. falciparum telomere repeat amplification protocol (Pf-TRAP) assay and Southern blot hybridization. Telomerase inhibitors such as 7-deaza-dGTP and AZT-TP, when used with the cytoplasmic extract of gametocytes in the Pf-TRAP assay, efficiently inhibit the product, which confirms the presence of telomerase in the gametocytes. The presence of telomerase activity in the laboratory adapted local (eastern India) isolates of P. falciparum indicates that telomerase might be the major player in chromosomal end protection during replication. The finding suggests that telomerase can be a potent target for the transmission blocking vaccine and drugs for combating malaria caused by P. falciparum.

Identification of a nuclear protein interacting with a novel site on rat androgen receptor promoter after transcription factor NFkB is displaced from adjacent site.

Sequence-specific DNA-protein interactions mediate the regulation of rat androgen receptor (rAR) gene expression. Previously, DNase I footprinting revealed that nuclear factor kappa B (NFkB) binds to region -574 to -554 on rAR promoter and represses its expression. In this study, we demonstrate that when NFkB protein is removed from its site by competitor DNA in DNase I footprinting reaction, a new DNase I protected region is formed overlapping adjacently (-594 to -561). This indicates that another nuclear protein (named here as FRN, factor repressed by NFkB) binds to rAR promoter only after NFkB protein is displaced. By competitive electrophoretic mobility shift assay and mutation analysis, we confirmed the formation of FRN-DNA complex. FRN interacts with a novel sequence on rAR promoter and may play a role in regulation of rAR gene expression in concert with NFkB.

Nuclear localization of senescence marker protein-30, SMP30, in cultured mouse hepatocytes and its similarity to RNA polymerase.

Senescence marker protein-30 (SMP30), expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity. By immunohistochemistry and western blotting, we found that SMP30 was in both the nuclei and cytoplasm of cultured mouse hepatocytes. By a homology search, we found that a domain of the SMP30 sequence 51 amino acid residues long was 60-66% similar to bacterial and yeast RNA polymerases.

Age-dependent transcription factor (ADF) interacts with prostate-specific antigen (PSA) gene enhancer.

Human prostate-specific antigen (PSA) is clinically most useful diagnostic marker for prostate cancer. The PSA gene is partially regulated by androgen hormone via androgen receptor (AR). Several transcription factors including novel transcriptional regulator, age-dependent factor (ADF) bind to AR promoter and play role in the regulation of AR gene expression. Earlier, an androgen responsive enhancer (-5824 to -3738) has been identified in 5'-flanking region of PSA gene. Here, we demonstrate by competitive electrophoretic mobility shift assay that ADF binds to a 19 bp sequence in PSA gene (-4372 to -4390) located within this enhancer region. This suggests that ADF may play a role in the regulation of PSA gene expression.