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Biomedical subjects

S Rajaraman

Publications and source records attributed to S Rajaraman.

At least 37 records · Page 2Linked to original sources

Desquamin is an epidermal ribonuclease.

Desquamin is a glycoprotein that we have isolated from the upper granular layer and the stratum corneum of human epidermis; it is not ordinarily expressed in submerged cultures, whose terminal differentiation stops short of formation of these layers. The exogenous addition of desquamin to human cultured keratinocytes extended their maturation, and hematoxylin staining indicated a loss of cell nuclei. For confirmation, cultured cells were lysed in situ, and the nuclei were incubated with desquamin for several days, then stained with hematoxylin. Damage to the nuclei was evident: the nuclear inclusions remained intact, while the surrounding basophilic nuclear matrix was degraded. Desquamin was then tested directly for nuclease activity. Ribonuclease activity was determined by incubating desquamin with human epidermal total RNA and monitoring the dose-dependent disappearance of the 28S and 18S ribosomal RNA bands in an agarose/formaldehyde gel. On RNA-containing zymogels, we confirmed the RNase activity to be specific to desquamin. Using synthetic RNA homopolymers, we found the active RNase domains to be limited to cytosine residues. On the contrary, DNA was not degraded by an analogous procedure, even after strand-separation by denaturation.

Cell Adhesion Molecules↗

Isoforms of cathepsin D and human epidermal differentiation.

Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.

Amino Acid Sequence↗

Diabetic nephropathy with anti-GBM nephritis.

Immune complex glomerulonephritis can be superimposed on diabetic glomerulosclerosis. Idiopathic membranous glomerulonephritis, immunoglobulin (Ig) A glomerulonephritis, Henoch-Schönlein nephritis, membranoproliferative glomerulonephritis, minimal change glomerulonephritis, postinfectious glomerulonephritis, lupus nephritis, amyloidosis, focal segmental glomerulosclerosis, and rarely crescentic glomerulonephritis can all occur with diabetic nephropathy. We describe for the first time an unusual case of diabetic nephropathy coexistent with anti-glomerular basement membrane (GBM) nephritis. The renal function of this patient improved with plasmapheresis and immunosuppressives. We also review the literature on coexistent rapidly progressive glomerulonephritis (RPGN) and diabetic nephropathy.

Anti-Glomerular Basement Membrane Disease↗

Acute renal failure in a patient with AIDS: histoplasmosis-induced granulomatous interstitial nephritis.

Although the most frequent cause of acute renal failure (ARF) in patients with AIDS is acute tubular necrosis (ATN) secondary to ischemic renal injury from septicemia, a spectrum of causes may result in ARF in these patients. We report a patient with AIDS who developed ARF and was found to have granulomatous interstitial nephritis as a result of disseminated histoplasmosis. Histoplasma capsulatum was seen in the interstitium of the kidney on renal biopsy. The patient was treated with amphotericin B and itraconazole. Although he continues to require hemodialysis 3 months after his initial presentation, his other presenting symptoms have resolved with antifungal therapy. We also discuss the literature on disseminated histoplasmosis and renal failure.

AIDS-Related Opportunistic Infections↗

Glutamine deprivation induces apoptosis in intestinal epithelial cells.

BACKGROUND: Glutamine is the most abundant amino acid in the blood, and its deprivation leads to gut mucosal atrophy. The small intestinal mucosa is maintained by a balance between cell proliferation and cell death by apoptosis. We reported that glutamine is required for nitrogen-stimulated proliferation in intestinal epithelial cells. We do not know whether glutamine regulates apoptosis in the gut. The purpose of this study is to determine whether glutamine deprivation induces apoptosis in rat intestinal epithelial (RIE-1) cells and to compare the effect of glutamine starvation with that of methionine and cysteine (Met/Cys) starvation. METHODS: RIE-1 cells were deprived of either glutamine or Met/Cys for 24 hours. Cell numbers were determined by cell counting and tetrazolium enzymatic assay. Apoptosis was quantified by Annexin V assay and confirmed by DNA gel electrophoresis and Hoecsht nuclear staining. RESULTS: Deprivation of glutamine or Met/Cys resulted in decreased cell numbers. However, only the glutamine-deprived group showed significant induction of apoptosis with increased Annexin V staining, DNA laddering, and nuclear condensation. CONCLUSIONS: This study provides biochemical and morphologic evidence that glutamine deprivation induces apoptosis in rat intestinal epithelial cells. In contrast, Met/Cys starvation suppresses cell number without induction of apoptosis. These results suggest that glutamine serves as a specific survival factor in enterocytes.

Animals↗

Detection and cloning of epidermal zinc-alpha 2-glycoprotein cDNA and expression in normal human skin and in tumors.

Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is almost ubiquitous in body fluids, and its antibody labels the corresponding secretory epithelia. We have found that Zn alpha 2gp is also expressed in human epidermis. We cloned the Zn alpha 2gp cDNA by screening our cDNA library, derived from epidermal keratinocytes, with a probe for prostate Zn alpha 2gp. It had complete nucleic acid sequence homology with that from prostate, including the signal peptide. Just as Zn alpha 2gp expression is higher in more differentiated breast tumors, so in skin tumors the highest mRNA levels occurred in the normal controls, the lowest in basal cell carcinomas (the least differentiated epidermal tumor type), and intermediate levels in squamous cell carcinomas and Merkel cell carcinomas. A similar increase in Zn alpha 2gp gene expression with differentiation was observed when epidermal keratinocytes were cultured in media that varied in cellular maturation potential.

Amino Acid Sequence↗

Immunocytochemical localization of C-myc and C-jun oncoproteins in hamster kidney and estrogen-induced kidney tumors.

The chronic administration of 17beta-estradiol to male Syrian hamsters for 6-7 months induces kidney tumors which express high levels of c-fos, c-myc and c-jun mRNA compared to surrounding tissue or untreated controls. In this study, we have investigated, by immunocytochemical methods, the cellular localization of c-myc and c-jun oncoproteins in estrogen-dependent kidney tumors, in kidney tissue of hamsters treated with 17beta-estradiol for 6 months and in the kidneys of age-matched controls. The c-myc oncoprotein was strongly expressed in tumors, in smooth muscle layers of arteries and in parietal epithelial cells of the glomerulus. In age-matched untreated kidneys, there was little or no staining in the glomerulus, arteries or kidney tubular cells. The c-jun oncoprotein was detected in kidney tumors and in the tubular epithelium of surrounding tissue. The immunoreactivity for c-jun oncoprotein was highest in the tumor, intermediate in estrogen-treated kidney tissue and lowest in kidney tubular cells of controls. It is concluded that the high expression of c-myc in estrogen-induced kidney tumors, in the smooth muscle layer of arteries, and in glomerular parietal epithelial cells in the kidneys of 17beta-estradiol-treated hamsters, but poor expression in control kidneys indicate an involvement of this oncoprotein in the tumorigenic process. In contrast, c-jun is expressed in untreated, in 17beta-estradiol-treated kidneys and in tumors, and may not serve as a prognostic marker in the transformation of these cells to the malignant phenotype.

Animals↗

Rat GST 8-8 is expressed predominantly in myeloid origin cells infiltrating the gravid uterus.

Previous studies from our laboratory have shown a relatively high expression of rGST8-8 in uterine tissues. This GST isozyme displays relatively high glutathione-peroxidase activity towards lipid-hydroperoxides and towards toxic 4-hydroxyalkenals generated from lipid peroxidation. Since the uterus is a unique organ, subject to oxidative stress due to infiltration by immune effector cells during gestation and because this infiltration is readily identifiable histologically, the studies reported herein were performed to localize the cell specific expression of rGST8-8 to determine whether immune effector cells infiltrating the pregnant rat uterus specifically expressed rGST8-8. A 75 bp end-radiolabeled cRNA probe was prepared from the full length mGSTA4-4 cDNA from the region which is highly homologous with rGST8-8. This cRNA probe was used for in situ hybridization studies to localize rGST8-8 in specific cell types of gravid rat uterus. Results of these studies indicate that this GST isozyme is selectively expressed in myeloid origin cells such as monocytes/macrophages, and neutrophils infiltrating the uterine endometrium and in vascular walls. Selective expression of rGST8-8 in the myeloid origin cells, which are known to generate higher levels of reactive oxygen species, suggests that this GST isozyme plays an important role in the protection mechanisms against lipid peroxidation.

Animals↗

Expression of MDR1 (multidrug resistance) gene and its protein in normal human kidney.

P-glycoprotein (Pgp), the product of the multidrug resistance (MDR) gene overexpressed in cancer cells, is present also in normal tissues. In the kidney, MDR1 Pgp has been found in the proximal tubule and in cultured mesangial cells. In situ hybridization and immunohistochemistry were used to determine the complete nephronal localization of MDR mRNA and its product, Pgp, in the human kidney. MDR mRNA expression was studied with the use of nonradioactive in situ MDR RNA probes. MDR1 Pgp was immunolocalized using the specific monoclonal antibody MRK16. The presence of MDR mRNA was confirmed in proximal tubules and mesangium, and demonstrated as well in thick limb of Henle's loops and in collecting ducts. MDR1 Pgp colocalized in the same nephronal segments. This suggests that, in addition to secreting xenobiotics, Pgp may play a role in the transport of endogenous substrates or in the regulation of Cl- channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

ICAM-1 expression in corneal epithelium of a patient with vernal keratoconjunctivitis: case report.

Recent reports have demonstrated the expression of intercellular adhesion molecule-1 (ICAM-1; CD54) on the epithelium in various allergic diseases and inflammatory conditions, including the bronchial epithelium of patients with allergic asthma, conjunctival epithelium of allergic patients after allergen-specific challenge, and corneal epithelium of rejected corneal allografts. We investigated the presence of ICAM-1 expression on the corneal epithelium from a patient with vernal keratoconjunctivitis (VKC). Immunohistochemical staining of the diseased cornea demonstrated abundant ICAM-1 expression on the corneal epithelium. Immunoreactive ICAM-1 appeared to localize primarily to the cells of the basal and middle layers of the corneal epithelium. No staining was detected on the ocular surface epithelium. The normal, healthy cornea demonstrated no significant ICAM-1 expression on any of the epithelial layers, similar to that previously reported. To the best of our knowledge, this is the first report of ICAM-1 expression on the corneal epithelium from a patient with VKC.

Adult↗

Prevention of graft-versus-host disease by intrathymic injection of recipient-type splenocytes into donor.

We have prevented graft-versus-host disease (GVHD) by tolerizing graft donors to host antigens by intrathymic injection of recipient-type splenocytes into donors. A unidirectional GVHD model was used in which intravenous injection of 3-4 x 10(8) Lewis rat (donor) lymphocytes into (Lewis x Brown Norway)F1 rats (recipients) causes lethal GVHD. The donor animals were divided into five treatment groups. The group 1 donor animals received no treatment. The group 2 donors received a single intraperitoneal injection of 1 ml of antilymphocyte antiserum (ALS). The group 3 donors received an intrathymic injection of 50x10(6) host splenocytes. The group 4 donors received both ALS (intraperitoneally) and intrathymic allograft. The group 5 donors received both ALS (intraperitoneally) and intravenous allograft. Two weeks after these treatments, 3-4x10(8) lymphocytes from each of these donors were injected (intravenously) into the recipients. The clinical signs of GVHD, as measured by profound weight loss, hair loss, inflammation of foot pads and ears, and profound splenomegaly, were evident in recipients of groups 1, 2, and 3 between days 9 and 10 and in the recipients (two of four) of group 5 on day 17. No GVHD was observed by histopathology in all 14 hosts that received lymphocyte injection from the group 4 donor animals (up to 300 days). These results demonstrate that GVHD can be eliminated by tolerizing donors toward host by intrathymic injection of the recipient-type lymphocytes into the donor. A single injection of ALS is necessary to possibly eliminate antihost response from the donor for the tolerance induction. The thymic route appears to be superior to the intravenous route for tolerance induction.

Adoptive Transfer↗

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Amino Acid Sequence↗

Gastrin gene expression is required for the proliferation and tumorigenicity of human colon cancer cells.

The majority of human colon cancers express the gastrin gene, and a significant percentage bind gastrin-like peptides. However, it is not known if gastrin gene products are physiologically relevant to the growth and proliferation of human colon cancers. To investigate the functional role of gastrin gene expression, we examined the effect of gastrin antisense (AS) RNA expression on the growth and tumorigenicity of colon cancer cells. The full-length human gastrin cDNA was cloned in the AS direction in a retroviral vector under the transcriptional control of human cytomegalovirus promoter. Three representative human colon cancer cell lines that expressed negligible (Colo-205A) to significant (Colo-320 and HCT-116) levels of gastrin mRNA were transfected with either AS or control vectors and subjected to various growth studies in vitro and in vivo. The proliferative and tumorigenic potential of the AS clones from the gastrin-expressing cell lines was significantly suppressed compared to that of the control clones, whereas the growth of Colo-205A-AS cells (the negative control) was similar to that of the Colo-205A-C-cells, indicating the relative specificity of the antitumorigenic effects of AS gastrin RNA expression. We believe that this is the first evidence that supports a possible critical role of gastrin gene expression in the tumorigenicity of human colon cancers that express the gastrin gene. Because > 60-80% of human colon cancers express the gastrin gene, it can be expected that the growth of a significant percentage of these cancers may be critically dependent on the expression of gastrin gene products. Therapeutic measures, such as the AS strategy used in the present study, may therefore prove to be useful in treating human colon cancers in the future.

Animals↗

Role of glutathione modulation in acrylonitrile-induced gastric DNA damage in rats.

Acrylonitrile (VCN) or its reactive metabolites irreversibly interact with gastric DNA in vivo and cause DNA damage. The effect of glutathione (GSH) modulation on VCN-induced genotoxicity and unscheduled DNA repair synthesis (UDRS) in DNA of gastric mucosal tissues was investigated. VCN-induced UDRS was determined: in control rats, rats with depleted gastric GSH contents, and rats treated with sulfhydryl compounds. A single oral dose (23 mg/kg) of VCN induced a time- and dose-dependent increase in gastric UDRS and decrease in GSH levels. While maximal UDRS in gastric mucosa was observed 2 h following oral administration of 23 mg/kg VCN, maximal GSH depletion (50% of control) was detected 4 h following treatment. Increasing the VCN dose to 46 mg/kg caused a further decrease in gastric GSH level (27% of control), while UDRS was elevated. Inhibition of VCN oxidation by treatment of the animals with the cytochrome P450 inhibitor, SKF 525-A, prior to VCN administration caused 65% reduction in VCN-induced UDRS. Treatment of rats with the GSH depletor diethylmaleate (DEM) prior to VCN administration caused 167% increase in UDRS in gastric mucosal tissues. Treatment of the animals with the sulfhydryl compounds, cysteine and penicillamine, prior to VCN administration protected against VCN-induced UDRS. The results demonstrated an inverse and highly significant correlation between gastric GSH levels and VCN-induced UDRS (r = -0.873, P < 0.0001). In conclusion, our study indicates that VCN bioactivation and the homeostasis of gastric GSH may play a major, role in the initial processes underlying VCN-induced gastric carcinogenesis.

Acrylonitrile↗

Fetal and neoplastic expression of the neurotensin gene in the human colon.

OBJECTIVE: The authors identified various colon cancers that express the gene for the gut peptide neurotensin (NT/N). In addition, the authors sought to delineate the temporal pattern of NT/N gene expression in the human fetal colon. SUMMARY BACKGROUND DATA: Expression of NT/N is localized to the mucosa of the adult small bowel but also has been identified in the fetal colon, which resembles the small bowel until the end of the second trimester. Ectopic NT/N expression has been shown in certain types of colon cancer, suggesting a reversion to a fetal phenotype. METHODS: Sensitive ribonuclease protection assays were used to determine NT/N expression in colon cancers and adjacent normal mucosa as well as colon cancers established as tumor xenografts and fetal colon samples. RESULTS: NT/N gene expression was shown in 4 of 12 (25%) human colon cancer xenografts and in 11 of 40 (28%) freshly resected colon adenocarcinomas; NT/N gene expression was not expressed in any of the samples of normal colonic mucosa adjacent to the tumors. The NT/N gene was expressed maximally in the fetal colon between 16 and 18 weeks' gestation; NT/N expression was decreased between 19 and 22 weeks and was not apparent in either the 24-week fetal colon or the adult samples. CONCLUSIONS: The NT/N gene expression is expressed transiently in the fetal colon during a development stage that is characterized by morphologic similarity to the small bowel. In addition, NT/N is reexpressed in approximately one fourth of the human colon cancers, indicating that neoplastic transformation leads to reversion to a fetal phenotype in certain types of colon cancer. The NT/N gene will provide a useful model to further define the complex differentiation pathways in the normal gut as well as the process of fetal "dedifferentiation" in certain types of colon cancer.

Adenocarcinoma↗

Recombinant desmoglein 3 has the necessary epitopes to adsorb and induce blister-causing antibodies.

The development of an animal model for studying the pathogenesis of pemphigus vulgaris (PV) has been hampered by the unavailability of the purified full-length autoantigen desmoglein 3 (Dsg 3).Therefore, we expressed Dsg 3 using a baculovirus expressed system. The expressed protein was identified as Dgs 3 by its reactivity with a pan-cadherin anti-serum, an anti-serum to a Dsg 3 synthetic peptide, or patient serum, and by amino-terminal sequencing. Carbohydrate analysis showed that recombinant Dsg 3 was glycosylated. While a majority of the recombinant protein was cell associated, by immunoprecipitation, some Dsg 3 was demonstrated in the medium. The Dgs 3 could adsorb out blister-causing antibodies from patient sera. Rabbit anti- Dsg 3 antibodies induced by the recombinant Dsg 3 showed specific binding to intercellular spaces of monkeys esophagus by indirect immunofluorescence. Moreover, these antibodies induced PV-like blisters in neonatal mice and weakly bound perilesional epidermis. Availability of large quantities of relatively pure Dsg 3 should now facilitate studies aimed at understanding Dsg 3 structure and pathogenesis of PV, with implications for developing specific immunotherapies.

Animals↗

Growth hormone upregulates gastrin and peptide YY gene expression.

The purpose of these studies was to examine the effects of excess growth hormone (GH) on gastrin and peptide YY (PYY) gene expression. Transgenic mice with the bovine GH gene linked to a mouse metallothionein I promoter were used as a model of chronic GH excess. Antral gastrin mRNA and peptide levels were elevated significantly (P < 0.05) in GH transgenic mice compared with wild type littermates. Ileal PYY mRNA and ileal and colonic PYY levels were significantly elevated in GH transgenic mice compared with wild type littermates. The elevations in gastrin and PYY gene expression in GH transgenic mice were independent of food intake. Serum concentrations of gastrin and PYY were also elevated in GH transgenic mice. Immunohistochemical analysis showed that the density of PYY-containing cells in the colon of GH transgenic mice and wild type littermates did not differ. In addition, the mRNA and protein levels of chromogranin A, a marker of endocrine cells, were not increased in the colon of GH transgenic mice. Together, these data indicate that GH, insulin-like growth factor I, or both can upregulate gastrointestinal gastrin and PYY gene expression directly.

Animals↗

Effect of endogenously produced parathyroid hormone-related peptide on growth of a human hepatoma cell line (Hep G2).

A well differentiated human hepatoma cell line (Hep G2) was used to explore potential roles for PTH-related peptide (PTHrP) as an autocrine/paracrine growth factor. Using Northern analysis or reverse transcription-PCR, Hep G2 cells were found to express messenger RNAs for both PTHrP and the cloned PTH/PTHrP receptor, and the cells exhibited specific binding for [125I]PTHrP(1-36). Hep G2 growth medium was found to contain relatively large amounts of immunoreactive PTHrP (30 vs. 1-2 pM in medium not exposed to cells), and the PTHrP in growth medium (conditioned medium) was shown to contain N-terminal PTHrP biological activity, as assessed by the ability of the medium to stimulate cAMP production in rat osteosarcoma cells (ROS 17/2.8). Conditioned medium produced a dose-dependent severalfold increase in ROS cell cAMP that could be blocked by the PTHrP receptor antagonist [Asn10,Leu11,DTrp12]PTHrP-(7-34). PTHrP in Hep G2 cells also was detected by immunocytochemistry using antiserum to either synthetic PTHrP-(1-34) or recombinant PTHrP-(-5 to 139). Furthermore, these antisera were found to inhibit the ability of PTHrP-(1-34) to stimulate ROS cell cAMP production. When either antiserum (1:800-1:100 dilution) was added to subconfluent Hep G2 cells in medium containing 5% FBS for 3 days, a dose-related 40-50% increase in cell number occurred that could be inhibited by concurrent addition of 10 microM synthetic PTHrP-(1-36). The results show that Hep G2 cells synthesize and secrete both immunoreactive and biologically active PTHrP. As neutralization of PTHrP secreted by these cells by the addition of antiserum to PTHrP resulted in increased cell growth, the findings suggest that PTHrP can function as an autocrine or paracrine growth factor to suppress the growth of these human hepatoma cells.

Animals↗