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Biomedical subjects

S K De

Publications and source records attributed to S K De.

At least 19 recordsLinked to original sources
PubMed1992

Hydrazine sulfate protection against endotoxin lethality: analysis of effects on expression of hepatic cytokine genes and an acute-phase gene.

Hydrazine sulfate (HS) pretreatment protects mice against the lethal effects of bacterial endotoxin lipopolysaccharide (LPS) through mechanisms yet to be established. The liver was examined as a model organ to determine HS effects on (a) LPS activation of leukocyte (Kupffer cell) interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) genes and (b) subsequent cytokine-mediated induction of the acute-phase response as measured by hepatic metallothionein (MT) gene expression. The utility of this model was documented by in situ hybridization which showed that acute induction by LPS of the IL-1 beta gene occurred in cells found in liver sinusoids, consistent with Kupffer cells, whereas induction of the MT gene occurred in hepatocytes. The cell specific expression of these genes was further verified by Northern blot hybridization to LPS-treated liver RNA which showed that the LPS-mediated increase in hepatic cytokine mRNA levels, unlike that of MT, was not prevented by D-galactosamine (D-GalN) treatment. Northern blot hybridization established that HS pretreatment did not block the acute induction of hepatic cytokine mRNAs (IL-1 beta and TNF-alpha) by LPS nor did it induce these cytokine mRNAs in the absence of LPS. Northern blot hybridization further established that HS did not prevent LPS-mediated activation of hepatocyte MT gene expression. Thus, HS does not prevent LPS from activating liver leukocytes. These results also suggest that HS pretreatment neither prevents the general release of cytokines from LPS activated leukocytes nor the general induction of acute-phase protein gene expression in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Proteins↗
PubMed1991

Metallothionein mRNA stability in chicken and mouse cells.

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.

Animals↗
PubMed1991

Metallothionein gene expression and metal regulation during preimplantation mouse embryo development (MT mRNA during early development).

In order to provide information concerning gene expression and regulation in the preimplantation mammalian embryo, and to explore the roles of metallothionein (MT) during this period of development, the constitutive and metal-induced MT mRNA levels in mouse ova, preimplantation embryos, and oviducts were determined. These results were correlated with the effects of transient exposure to high levels of metals (zinc (Zn) or cadmium (Cd] on the continued development of preimplantation embryos into blastocysts in culture. RNA from preimplantation mouse embryos at different stages of development (Days 1 through 4 of gestation; D1 = vaginal plug) was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) to specifically amplify MT-I and MT-II mRNA transcripts. MT-I mRNA in ova, preimplantation embryos, and oviducts was detected using in situ hybridization. This mRNA in the oviduct was also analyzed by Northern blotting. The results establish that the mouse MT genes are coordinately and constitutively expressed at low basal levels in ova and preimplantation mouse embryos. In unfertilized (ova), fertilized (one-cell) eggs, and two-cell embryos, the MT-I gene was not detectably responsive to metal ions, whereas in later cleavage stage embryos (four- and eight-cell) the MT-I gene was detectably responsive to metals in some blastomeres of some of the embryos. In contrast, after the third cleavage this gene was highly metal-inducible in essentially all cells of the embryo (morula/blastocyst). Surprisingly, the appearance of metal responsiveness of the MT genes during development correlated with decreased Zn toxicity and increased Cd toxicity; two-cell embryos were Zn-sensitive and Cd-resistant, whereas eight-cell and older embryos were Zn-resistant and Cd-sensitive. In the oviduct, MT-I mRNA was not abundant in total RNA, but was detected specifically in the epithelial cells of the isthmus region and was elevated in these cells on D3 and D4 of gestation. In the oviduct, only isthmus epithelial cells responded to metals (Zn or Cd) by increased accumulation of this mRNA. These studies suggest that preimplantation mouse embryo develops the capacity to respond to metals in the environmental milieu by induction of MT gene expression at about the third cleavage. Whether the lack of responsiveness of these genes before this stage reflects transcriptional repression or attenuated metal ion influx and/or enhanced efflux remains to be determined. Sensitivity and resistance of preimplantation embryos to acute metal toxicity involve mechanisms other than MT gene expression in preimplantation mouse embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

High levels of metallothionein messenger RNAs in male germ cells of the adult mouse.

Northern blotting, in situ hybridization, and oligodeoxyribonucleotide excess solution hybridization were used to quantitate metallothionein-I (MT-I) and MT-II mRNAs in mouse testes. Testes from sexually mature adults contained high levels of both MT mRNAs (approximately 10-fold higher than those in control adult liver). Testicular MT mRNA levels were age dependent, being low the first 2 weeks after birth and increasing slowly thereafter to maximal levels in the adult (by 9 weeks after birth). In the adult testis, in situ hybridization indicated that only cells within the adluminal compartment (germ cells) of the seminiferous tubules contain high levels of MT mRNA. The appearance of cells containing elevated levels of MT mRNA during development was delayed from the onset of spermatogenesis. In situ hybridization suggested that MT mRNA accumulates after the initial differentiation of primary spermatocytes and is maintained in spermatids. Pachytene spermatocytes (PSC) and round spermatids (RTD) isolated from adult testes contained both MT-I and MT-II mRNAs in levels equivalent to those found in zinc-treated hepatocytes, whereas very low levels of MT mRNA were detected in isolated Sertoli cells (ST). In situ hybridization suggested that MT mRNA was present at only basal levels in interstitial, spermatogonial, and mature sperm cells at all developmental stages examined. Northern blot and in situ hybridization to sulfated glycoprotein-2 (SGP-2) mRNA, a ST-specific transcript, showed that SGP-2 mRNA is high in the testis of 1-week-old mice and decreases gradually to a lower level in the adult. In situ detection of this mRNA was consistent with the location of ST in the testis. SGP-2 mRNA was abundant in ST and rare in PSC and RTD preparations. Analysis of pulse-labeled proteins from isolated PSC and RTD indicated that these cells actively synthesize MT-I and MT-II. The high levels of MT mRNA in adult testes were not increased substantially after systemic injection of cadmium, zinc, or bacterial lipopolysaccharide. In marked contrast, these treatments led to dramatically increased levels of hepatic and ovarian MT mRNA. This study establishes that the MT genes are actively expressed in a developmentally regulated fashion in the male germ cells of the mouse. This suggests a role for MT in the process of spermatogenesis.

Animals↗
PubMed1990

Endotoxin induction of murine metallothionein gene expression.

Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (IL-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha). In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-20-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and MT mRNA. In each organ examined, LPS, IL-1 alpha, or IL-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver. IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

Animals↗
PubMed1990

Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos.

As an approach toward understanding the mechanisms by which cadmium (Cd) exerts its teratogenic effects, the expression and metal regulation of the metallothionein (MT) genes in midgestation mouse embryos were studied by Northern blot and in situ hybridization. Maternal injection of a teratogenic dosage of Cd (50 mumol Cd/kg body wt) did not induce MT mRNA in day 10 (D10) CD-1 mouse embryos, whereas zinc (Zn) (50 mumol/kg was an effective inducer. In contrast, Cd was about 10-fold more potent than Zn at rapidly inducing MT mRNA in D10 embryos incubated in vitro in medium containing micromolar concentrations of these metals. This suggests that following maternal injection, Cd but not Zn is prevented from reaching the D10 embryo and establishes that the embryonic MT genes are not refractory to metal induction, which might have explained the sensitivity of the embryo to Cd. MT mRNA was detected at high levels only in the extraembryonic membranes of D9 embryos exposed to Cd in vivo. On days 9 and 10, no embryonic cell types contained detectable levels of MT mRNA. This mRNA was detected first at low levels in hepatocytes on D11, soon after formation of liver and these levels increased dramatically by D12. Therefore, Cd teratogenicity was not associated with high levels of cell type-specific expression of the MT genes in Cd-sensitive regions of the embryo (neural tube, limb bud), that might have served to target Cd to these cells. Taken together, the results of this study suggest that Cd teratogenicity reflects damage to maternal or extraembryonic tissues. However, the results cannot exclude the possibility that certain cells in the embryo are exceptionally sensitive to low levels of Cd.

Abnormalities, Drug-Induced↗
PubMed1990

Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells.

Immunocytochemical analyses, using several mouse epidermal growth factor (EGF) polyclonal antibodies, detected immunoreactivity only in uterine luminal and glandular epithelia on late proestrus, estrus, and early on day 1 of pregnancy, but not late on day 1. This immunoreactivity was not detected in the ovariectomized uterus, but after estrogen stimulation it was detected first in the luminal epithelium between 12-24 h and then also in the glandular epithelium by 48 h. After 72 h of estrogen withdrawal, EGF immunoreactivity was no longer detected. This response was specific for estrogen and did not occur after progesterone injection (2 mg/day for 4 days). Using antipeptide antibodies specific for prepro-EGF, no immunoreactivity was detected in the ovariectomized uterus, weak reactivity was detected in the estrogenized uterus and submandibular gland, and strong reactivity was detected in the kidney. Northern blot analysis of uterine RNA failed to detect the expected 4.8-kilobase prepro-EGF mRNA, but, instead, a rare transcript of 2.4 kilobases was detected, which suggests that EGF mRNA is alternately processed in the uterus. The presence of an EGF-coding uterine transcript was further documented by hybridization of an EGF-coding region-specific oligodeoxyribonucleotide (oligo) to polymerase chain reaction-amplified uterine cDNA. In situ hybridization, using a prepro-EGF cRNA probe as well as an EGF-coding region-specific oligo, showed hybridization that colocalized with the EGF immunostaining (epithelia) and was absent from non-EGF-immunoreactive cells. Pulse labeling experiments coupled with immunoaffinity chromatography showed that estrogen induced an increase in the relative rate of synthesis of an acid-soluble immunoreactive protein which was the same size as authentic EGF. Furthermore, analysis of acid-soluble uterine proteins fractionated by DEAE-cellulose chromatography demonstrated a single coincident peak of antigenic activity and receptor-binding activity which coeluted from the column with authentic EGF. Electron microscopy localized EGF immunoreactivity to the Golgi of luminal epithelial cells. Taken together these results suggest that estrogen regulates expression of the EGF gene specifically in uterine epithelial cells. Increased expression of this gene results in an increase in the relative rate of synthesis of this protein and the accumulation of mature EGF.

Animals↗
PubMed1989

Unusual presentation of neurilemmoma in the cerebellopontine angle.

An unusual presentation of a neurilemmoma in the cerebellopontine (CP) angle is reported. The tumour was localised in the CP angle and the case presented with post-auricular swelling and erosion of the squamous part of temporal bone without involvement of the auditory or other cranial nerves.

Adult↗
PubMed1989

Cell-specific metallothionein gene expression in mouse decidua and placentae.

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.

Animals↗
PubMed1988

Tumours of the mastoid temporal bone: with interesting cases in the paediatric age group.

Tumours of the mastoid temporal bone are interesting clinical entities owing to their rarity. A wide variety of benign and malignant tumours occur in and around the temporal bone reflecting the complicated embryological origin and resultant diversity of tumours found in the area. Friedmann (1974), in a classification of tumours of the ear, has grouped some tumours as intermediate in variety, e.g. glomus jugulare tumour, in addition to various benign and malignant tumours. Metastatic deposits from various distant organs such as the lung, breast and kidney can occur in the mastoid bone (Schucknecht, 1968). Invasive tumours can spread to the mastoid bone from adjacent areas such as neoplasms of the parotid and cranium.

Child↗
PubMed1988

Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells.

Rat trophoblast tissue was found to synthesize a number of low molecular weight proteins possessing prolactin-like characteristics. There appear to be at least three proteins that cross-react with antisera to pituitary prolactin. Two of the proteins had a molecular weight of 25,000, similar to ovine pituitary prolactin, and isoelectric points of 6.8 and 7.0. The third immunoreactive protein had a lower molecular weight (23,500), similar in size to human placental lactogen, and a slightly more acidic isoelectric point of 6.75. The molecular weight variants cross-reacted with an antipeptide serum that was generated to a synthetic peptide representing amino acids 150 to 164 of rat placental lactogen-2 (PL-2). Based on this analysis, we consider these proteins to be related to PL-2. Analysis of trophoblast proteins by gel-filtration chromatography resulted in the identification of another trophoblast prolactin. This material eluted earlier than PL-2-related proteins on a gel-filtration column, possessed prolactin-like activity (determined by competition with ovine pituitary prolactin for rabbit mammary gland or rat liver prolactin receptors) but showed limited cross-reactivity with either the antiserum to pituitary prolactin or the antiserum to the PL-2 peptide. We have thus identified multiple low molecular weight trophoblast prolactins, possessing different biochemical and immunological characteristics.

Animals↗
PubMed1987

Establishment of a rat placental cell line expressing characteristics of extraembryonic membranes.

A cell line was derived from midgestation chorioallantoic placental explants of the outbred Holtzman rat. The cell line was found to express characteristics of extraembryonic membranes and to grow when introduced into allogeneic hosts. Growth in allogeneic hosts was detected following intraperitoneal injection of the cells but not following subcutaneous injection. The transplanted cells grew as cystic structures free in the peritoneum and as solid masses adhered to various abdominal organs. Cystic structures had a homogeneous morphology consisting of an epithelial-like cell layer surrounding a fluid-filled sac. Solid masses had a heterogeneous morphology, containing parts resembling normal components of the extraembryonic membranes (trophoblast, parietal, and visceral yolk sacs). Biochemical analysis of the placenta-derived cell line and transplanted structures derived from the cell line indicated that the cells had the potential to produce a variety of proteins characteristic of extraembryonic tissues. Cultured cells and both types of in vivo transplants produced the basement membrane protein, laminin. Peritoneal cystic structures also contained alpha-fetoprotein mRNA and very high levels of c-fos mRNA. Solid masses demonstrated elevated alkaline phosphatase activity, a marker of trophoblast cells. Cells grown in vitro expressed elevated c-myc mRNA levels, whereas, c-myc mRNA levels were reduced in the in vivo transplants. The behavior of the cell line in vitro and following in vivo transplantation suggests it contains elements capable of differentiation toward various components of the extraembryonic membranes. The results indicate that the rat placental cell line will be valuable for future studies on the differentiation of trophoblast cells and other components of the extraembryonic membranes.

Alkaline Phosphatase↗
PubMed1986

Purification, characterization and origin of rat gastric peroxidase.

A membrane-bound peroxidase (EC 1.11.1.7) from rat stomach has been solubilized by 0.2% cetyltrimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355-fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of Mr 45,000. In gel permeation, the Mr has been estimated as 47,000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN- and to 430 nm on reduction with dithionite. The velocity constant, k1 for the reaction of the peroxidase with H2O2 is 1.38 X 10(7) M-1 s-1 and Km for H2O2 is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p-hydroxymercuribenzoate is more reactive than mersalyl or N-ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65 degrees C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells.

Animals↗
PubMed1986

Horseradish peroxidase-catalyzed conversion of iodine to iodide in presence of EDTA and H2O2.

EDTA (4 mM) blocks the oxidation of iodide to I-3 (increase of extinction at 353 nm) by H2O2 catalyzed by horseradish peroxidase, which is reversed by the addition of an equimolar concentration of Zn2+. Addition of suboptimal concentration of EDTA (2 mM) not only decreases the rate of forward reaction of I-3 formation but also causes loss of extinction of the same when I-3 is generated. The loss of extinction of I-3 is proportional to the enzyme concentration and is blocked by azide, the inhibitor of the peroxidase. EDTA also causes bleaching of nonenzymatically formed I-3 (from iodide and H2O2) only in the presence of horseradish peroxidase, and the effect is reversed by the equimolar concentration of Zn2+. Both the bleaching of I-3 by EDTA and reversal of EDTA effect by Zn2+ are sensitive to azide. The decrease of extinction of I-3 (formed by dissolving iodine in KI solution) is dependent on EDTA, H2O2, and horseradish peroxidase. Molecular iodine is also bleached but at a slower rate than I-3. Evidence is presented to show that this bleaching of I-3 is due to enzymatic conversion of I-3 to iodide in presence of EDTA and H2O2 and this involves pseudocatalatic degradation of H2O2 to O2.

Edetic Acid↗
PubMed1986

Localization and origin of the intestinal peroxidase--effect of adrenal glucocorticoids.

Peroxidase activity in rat intestine is stimulated two-fold after bilateral adrenalectomy and is reversed by dexamethasone (9-fluoro-11 beta,17,21-trihydroxy-16 alpha-methyl-1-4-pregnadiene-3,20-dione). The enzyme activity is inhibited on administration of various glucocorticoids of which dexamethasone acts as the most potent inhibitor of the enzyme in vivo. The change of enzyme activity results neither from alteration of the apparent Km of the enzyme nor from enzyme synthesis. Although a small amount of peroxidase is located in the intestinal epithelial cells, a large amount is present in the rest of the intestine. Histochemical studies indicate the presence of peroxidase in the lamina propria, the core of the intestinal villi which contains eosinophil. The peroxidase isolated from the epithelial cell-free intestine is similar to the peroxidase obtained from the pure eosinophil in terms of various physicochemical properties. Dexamethasone also inhibits the eosinophil peroxidase and decreases the number of both circulating and intestinal eosinophil. Studies indicate that a large part of the peroxidase of the intestine is contributed by invading eosinophil and dexamethasone inhibits the enzyme by sequestration of eosinophil both from intestine and blood possibly to the peripheral lymph nodes.

Adrenal Glands↗