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Biomedical subjects

S S Chauhan

Publications and source records attributed to S S Chauhan.

At least 19 recordsLinked to original sources
PubMed2002

Differential activity of cathepsin L in human placenta at two different stages of gestation.

The implantation of blastocyst depends on the invasiveness of the syncytiotrophoblast, which penetrates the maternal decidua to establish the placenta. Cathepsin L, a lysosomal cysteine protease over-expressed in a variety of human malignancies, has been implicated in tumour invasion and metastasis. Specific inhibitors of cathepsin L inhibit the invasion of amnion by murine tumour cells. Previous studies have revealed that tumour and trophoblast invasiveness are mediated by shared factors, like metalloproteinases and laminin. Several proteases closely related to cathepsin L have recently been reported in the placentae of different species. In the present study, we demonstrate the expression of cathepsin L in human placenta by immunohistochemical analysis and RT-PCR followed by Southern hybridization. The activity of cathepsin L against the synthetic dipeptidyl substrate, Cbz-Phe-Arg-N-Methylcoumarin, was assayed. E-64, a specific inhibitor of cathepsin L was used to confirm that the enzyme activity being measured was due to cathepsin L. We observed the specific activity of cathepsin L in first trimester placenta to be significantly higher as compared to the term placenta. However, the levels of placental cathepsin L mRNA were comparable at these two stages of gestation. The increased enzymatic activity of cathepsin L in the invasive phase of placentation taken together with its previously established role in tumour invasion and metastasis indicates the involvement of this protease in trophoblast invasion.

Adult↗
PubMed2001

Cloning and characterization of human cathepsin L promoter.

Cathepsin L is a lysosomal cysteine protease, which is over-expressed and secreted by malignant cells. It is very potent in degrading collagen, elastin, laminin and other components of the basement membrane and, therefore, has been implicated in tumor invasion and metastasis. The structural portion of the human cathepsin L (hCATL) gene was cloned to elucidate its genomic organization (Chauhan et al., J. Biol. Chem. 218 (1993) 1039). In the present study, a 1.90 kb DNA fragment, containing 1825 bp of the 5' upstream region of hCATL and 75 bases of the first exon of the hCATL, was amplified by PCR from an adaptor ligated placental genomic library. This fragment has been demonstrated to exhibit promoter activity by luciferase reporter assays. Sequence analysis of this fragment revealed the presence of approximately 29 different putative transcription factor binding sites. Several of them like AP-4, GATA-1, Lmo2, CEBPB, MZF-1, NF-AT, etc. were present more than once in this region. However, a consensus CAAT box but no consensus TATA box was found within the 1.0 kb upstream of exon 1. The transcription initiation site of hCATL, using placental total RNA, was mapped to a single adenine residue 289 bases upstream of the ATG codon.

5' Flanking Region↗
PubMed1998

Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L.

Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells. However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear. We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization. High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium. At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes. Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium. Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution. Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion. Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT L, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium. We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.

3T3 Cells↗
PubMed1998

Exercise-induced ventricular arrhythmias in congestive heart failure and role of ACE inhibitors.

Ventricular arrhythmias are considered to be related to left ventricular (LV) dysfunction. ACE inhibitors though improve LV function their beneficial role on exercise-induced ventricular arrhythmias is not established. To study the effects of ACE inhibitors on exercise capacity vis-a-vis their role on exercise-induced ventricular arrhythmias, 25 patients of congestive heart failure (CHF) of various etiologies in NYHA Class II and III were subjected to a prospective randomised controlled trial. The control group comprising of 12 patients received conventional treatment (digitalis and diuretics) and the test group was given enalapril/captopril in addition as tolerated. They were followed up for 3 months. Exercise testing on treadmill and monitoring of clinical and biochemical parameters were done at the beginning and end of study in all cases. Ventricular arrhythmias observed during exercise and post-exercise for 10 minutes was analysed using Lown's grading for frequency and severity of ventricular arrhythmia. The mean exercise duration showed significant improvement on ACE inhibitor as compared to the control group (p < 0.05) however there was no significant change in the grades of arrhythmia. Serum electrolytes and other bio-chemical parameter were within normal range. It is concluded that effect of ACE inhibitor on improving functional capacity in CHF is independent of it's any effect on exercise-induced ventricular arrhythmias.

Adult↗
PubMed1998

The changed clinical spectrum of malaria due to drug resistance.

The clinical spectrum of 14 cases of Plasmodium falciparum malaria (PF) who received empirical treatment and suffered from initial prolonged mild illness culminating into severe complicated malaria are presented. The empirical treatment (ET) consisted of adequate doses of chloroquine in 9, chloroquine with pyrimethamine-sulphadoxine combination in 3 and pyrimethamine-sulphadoxine alone in 2 cases. Moderate fever and weakness persisted for 7 to 28 days leading to anaemia and progressive hepatosplenomegaly in all patients. Other clinical features noticed included jaundice in 5, sudden shock with pulmonary oedema in 4, cerebral malaria and renal failure in 3 each and multiorgan in 4 cases. Subsequent investigations revealed PF rings in 9 cases, mixed PF and vivax infection in 3 and PF gametocytaemia only in 2 patients. Seven patients received quinine, 4 quinine with doxycycline and 3 were given quinine followed by injection artemether. Exchange transfusion was carried out in two cases. Four patients died. The empirical treatment with first line antimalarials alters the clinical profile of resistant PF, makes it milder temporarily, delays in confirming the diagnosis and leads to high mortality. There is urgent need for more diligent early workup for these patients who linger on with moderate pyrexia, progressive hepatosplenomegaly, anaemia and jaundice after ET till better diagnostic methods are available to avoid the prolonged illness and high mortality.

Adolescent↗
PubMed1997

Evaluation of P-glycoprotein expression in human oral oncogenesis: correlation with clinicopathological features.

To determine whether the multidrug-resistance-gene product phospho-glycoprotein (P-gp) is implicated in progression of oral tumours and/or drug resistance, the expression of P-gp was examined in different stages of oral oncogenesis using monoclonal antibody C-219. Cryosections from normal (41 cases), dysplastic lesions (32 cases), untreated primary SCCs (50 cases) and recurrent tumours (31 cases) were used for immunostaining, and the results were corroborated by immunoblotting. Chi-square test for trend analysis showed a significant increase in P-gp immunopositivity across the normal, leukoplakia, primary oral SCC and recurrent SCC groups (p < 0.01). Expression of P-gp in dysplastic lesions showed significant association with severity of dysplasia, the level of P-gp protein being higher in severe and moderate dysplasia. Among the primary tumours, significant correlation was observed between P-gp positivity as well as level of P-gp expression and tumour stage. The recurrent tumours showed significant increase in P-gp expression as compared with untreated primary oral tumours. We conclude that differential expression of P-gp may be an index of the disease prognosis in oral-cancer patients in the context of the Indian population.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Role of proteases in tumor invasion and metastasis.

Cancer remains a major cause of worldwide deaths due to ability of cancer cells to form secondary tumors at other sites by multistep process called metastasis. In order to migrate from their original site, tumor cells have to cross several barriers like basement membranes, interstitial tissues and extracellular matrices, which are composed primarily of collagen, proteoglycans, elastin, laminin and other glycoproteins. Tumor cells over express and secrete proteases which are capable of degrading the components of these barriers and thus facilitate their migration. The classes of proteases which have been implicated in the process of tumor invasion and metastasis include metalloproteases, serine proteases and cathepsins. Cancer cells in general have elevated levels of proteases belonging to more than one class. In some studies, process of invasion has been inhibited by using specific inhibitors of these proteases. Expression of some proteases has been observed only in some specific tumors. These proteases have been proposed to be of diagnostic/prognostic value. However a better understanding of the process of metastasis and tumor invasion is required before proteases can be used as therapeutic targets for blocking the spread of cancer.

Animals↗
PubMed1995

Effect of Japanese mint (Mentha arvensis) oil as fumigant on nutritional quality of stored sorghum.

Japanese mint (Mentha arvensis) oil (JMO) can be used effectively as fumigant against Sitophilus oryzae in stored sorghum. The effect of JMO at a dose of 166 microliter/l of space on nutrient composition and protein quality was studied in infested and uninfested sorghum grains stored for 3 months. The results revealed non significant effect of JMO on gran moisture, total ash, crude fibre, crude fat, crude protein and fat acidity in infested and uninfested grains at the end of 3 months storage. The JMO treatment had small but significant effect on reducing and non-reducing sugars. The values of Protein Efficiency Ratio (PER) for uninfested JMO treated grains, infested JMO treated grains and for untreated control stored for 3 months were 1.11, 1.07 and 1.09, respectively against control casein diet for which it was 2.15.

Dietary Carbohydrates↗
PubMed1993

Cloning, genomic organization, and chromosomal localization of human cathepsin L.

Cathepsin L is a lysosomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors, and tumor promoters. Many human tumors express high levels of cathepsin L, which is a broad spectrum protease with potent elastase and collagenase activities. Two published human cathepsin L cDNA sequences differ only in their 5'-untranslated regions. In this study, we demonstrate the concurrent expression of two distinct human cathepsin L mRNAs (hCATL-A and hCATL-B) in adenocarcinoma, hepatoma, and renal cancer cell lines. Cloning of the human cathepsin L gene by polymerase chain reaction amplification of genomic DNA and subsequent sequencing reveals that hCATL-A and hCATL-B mRNAs are encoded by a single gene. The 3' end of the first intron contains the 5' portion of hCATL-B and is contiguous to the second exon of the gene. These data suggest either the possibility of alternative splicing or the presence of a second promoter within the first intron of the hCATL gene. We mapped the hCATL gene to chromosome 9q21-22. Sequencing of both the mouse and human cathepsin L genes demonstrates almost complete conservation of exon and intron position, but significant divergence in intron structure, possibly reflecting differences in regulation of expression of the mouse and human cathepsin L genes.

Amino Acid Sequence↗
PubMed1992

Construction of a new universal vector for insertional mutagenesis by homologous recombination.

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neoR) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neoR are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neoR and tk cassettes are separated by four unique cloning sites on the right-hand side of the neoR cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.

Animals↗
PubMed1992

Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Expression of cathepsin L in human tumors.

It has been proposed that proteases secreted by cancer cells facilitate tumor invasion and metastasis by degrading the components of extracellular membranes. The lysosomal cysteine protease cathepsin L is synthesized in large amounts and secreted by many malignantly transformed cells in culture. The secreted protease is potent in degrading collagen, laminin, elastin, and other structural proteins of basement membranes. To determine whether human cancers synthesize cathepsin L, the expression of cathepsin L in approximately 100 human tumor samples was determined by quantitative RNA slot blot analysis using a specific human cathepsin L complementary DNA probe. Results of the present study suggest that cancers in general express higher levels of cathepsin L than do normal tissues. Kidney and testicular tumors expressed the highest levels of cathepsin L; non-small cell carcinomas of the lung expressed the next highest levels; and most cancers of the breast, ovary, colon, adrenal, bladder, prostate, and thyroid expressed elevated levels as well. Cathepsin L may prove useful as a diagnostic or prognostic marker of human malignancy.

Cathepsin L↗
PubMed1991

Elevation of rat pulmonary, hepatic and lung surfactant lipids by fly ash inhalation.

Fly ash contains many polycyclic aromatic hydrocarbons and genotoxic trace elements. In rats, fly ash exposure profoundly affects lung and liver histology. In the present study, the effect of fly ash inhalation on lung and liver lipids of rats was examined. Male Wistar strain rats were exposed daily to fly ash (0.27 +/- 0.01 mg/L air) in an inhalation chamber, 6 hr daily over a period of 15 days, and were killed on various days, i.e. 16, 30, 60, and 120. Fly ash inhalation significantly (P less than 0.05) increased total phospholipids (PL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in lungs. PC and dipalmitoylphosphatidylcholine (DPPC) contents in microsomes and lung surfactant also were significantly (P less than 0.05) higher in rats exposed to fly ash compared to control group animals. Radiolabeled precursor incorporation studies indicated that fly ash induced the synthesis of PC and DPPC by both CDP-choline pathway and N-methylation of PE in lung microsomes and enhanced their secretion into lung surfactant. In liver, PC and PE contents were elevated significantly (P less than 0.05) by fly ash exposure on days 16 and 30 respectively. A similar elevation of PC was observed in hepatic microsomes; this increase was due to its increased synthesis. However, the increased synthesis of PC in liver occurred to a greater extent by the N-methylation pathway than by the CDP-choline pathway.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Downstream sequences mediate induction of the mouse cathepsin L promoter by phorbol esters.

The major excreted protein (MEP) of mouse fibroblasts is the precursor to a lysosomal acid protease (cathepsin L) whose synthesis is induced by malignant transformation, growth factors, tumor promoters, and cyclic AMP. We have previously cloned a functional gene for MEP from NIH 3T3 cells. When subcloned into chloramphenicol acetyl transferase (CAT) expression vectors, both 4-kilobase and 300 base pair fragments in the 5'-flanking region of the MEP gene confer CAT activity that is stimulated by cyclic AMP treatment but is not stimulated by phorbol ester treatment of NIH 3T3 cells. These fragments confer constitutive promoter activity that is comparable to that of the SV40 promoter. Primer extension, using RNA from cells transiently transfected with MEP-CAT fusion plasmids, demonstrates that phorbol ester treatment increases the amount of transcript from constructs containing both the promoter and sequences downstream of the transcription initiation site, including the first three introns, but not from constructs containing only the 5'-flanking region of the MEP gene. Nuclear run-off experiments confirm that the increase in endogenous MEP mRNA is mediated by increased transcription and not via relief of transcriptional attenuation. Since both the MEP promoter, which contains three potential binding sites for the AP-2 transcription factor, and the SV40 promoter, which contains both AP-1 and AP-2 binding sites, fail to respond to 12-O-tetradecanoylphorbol-13-acetate in NIH 3T3 cells, these upstream motifs are not sufficient to confer phorbol ester responsiveness in NIH 3T3 cells. These results suggest that the MEP gene is regulated in a complex manner by sequences both upstream and downstream of the transcription initiation site.

Animals↗
PubMed1990

Adult subacute mountain sickness--a syndrome of congestive heart failure in man at very high altitude.

A new type of mountain sickness is described. 21 men (age 22.2, standard deviation [SD] 1.8 years) had severe congestive heart failure with oedema and ascites after 10.8 (SD 5.9) weeks at altitudes of 5800-6700 m. Investigation, within 3 days of transfer to 300 m, showed polycythaemia, cardiomegaly with right ventricular enlargement, and (in 17) pericardial effusion. The heart failure resolved rapidly after transfer from high altitude.

Acute Disease↗
PubMed1990

Placental transfer of metals of coal fly ash into various fetal organs of rat.

Fly ash (100 mg/kg body weight) was administered intratracheally to 14-day pregnant rats for 6 consecutive days. On day 20 of gestation the translocation of metals present in the fly ash to various maternal and fetal organs was studied. Fly ash administration to pregnant mothers retarded the growth of fetal heart and kidney as determined by their weights. Fly ash instillation increased organ levels of nearly all the metals studied in both mother and fetus. Most of the metals present in coal fly ash were transferred in significant amounts through placenta to several fetal organs. However, the pattern of their distribution into various fetal organs was different for different metals.

Animals↗