Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.     

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.     

Lack of correspondence between mRNA expression for a putative cell death molecule (SGP-2) and neuronal cell death in the central nervous system

Neuronal death during nervous system development, a widely observed phenomenon, occurs through unknown mechanisms. Recent evidence suggests an active, destructive process requiring new gene expression. Sulfated glycoprotein-2 (SGP-2), a secretory product of testicular Sertoli cells has been shown to up-regulate in several nonneural tissues undergoing programmed cell death and in several types of neuronal degeneration. In order to determine if this message up-regulates in neurons undergoing developmentally determined cell death, we have studied the expression of SGP-2 mRNA in the developing and adult rat central nervous system (CNS) with in situ hybridization. We also report on the expression of this message in nonneural tissues from several regions of the developing embryo. The developing and adult rat central nervous system as well as widely varied tissues in the rat embryo express SGP-2 mRNA in a pattern that does not correlate with regions undergoing developmental cell death. In the nervous system, SGP-2 mRNA is expressed in neuronal populations including motor neurons, cortical neurons, and hypothalamic neurons at ages when the period of developmental cell death has passed. In a nonneural tissue (palatal shelve epithelium) for which a developmental cell death period has been described, SGP-2 mRNA was not present in the region where cell death occurs. We conclude that SGP-2 mRNA expression cannot be correlated with programmed cell death in neural or nonneural tissues. The results of this study as well as recently reported SGP-2 homologies indicate a possible role for this protein in secretion and lipid transport.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

The patterns and expression of KDR in normal tissues of human internal organs

KDR has been implicated for playing an important role in the formation of new blood vessels and in solid tumor growth. It was considered as one of the most important regulators of angiogenesis and a key target in anticancer treatment. In the present study, we characterized KDR mRNA and protein expression in normal tissues of perinatal and adult tissues using One-step Real-Time RT-PCR and immunohistochemistry with a self-made anti-KDR antibody. The expression of KDR mRNA and protein in perinatal internal organs were all higher than in adult organs including brain, kidney, liver, lung and heart, respectively. KDR protein was presented in the cell plasma membrane of human internal tissues. The expression of KDR protein was raised in macrophage of spleen, and decreased in neurons of brain, myocardium, bronchial epithelial cells and alveolar epithelial cell, proximal and distal tubules cells, and hepatic cells with the maturity process of human organs. Notably, the order of KDR protein expression from highest to lowest is as follows: brain, liver, heart, kidney, and lung in adult tissues with statistically significant. It follows that how to balance the potential therapeutic side effect with human internal organs in targeted therapy of over-expressing KDR tumor.     

Induction of the synthesis of the C-X-C chemokine interferon-γ-inducible protein-10 in experimental canine endotoxemia

Endotoxemia is associated with a systemic inflammatory response leading to organ-specific leukocyte recruitment and tissue injury. Chemokine expression has been demonstrated in various models of sepsis and may mediate tissue infiltration with inflammatory cells. In this study we examined expression of the C-X-C chemokine interferon-gamma-inducible protein-10 (IP-10), a potent T-lymphocyte chemoattractant, in a canine model of endotoxemia and investigated mechanisms of cytokine-mediated IP-10 induction in endothelial cells. Control canine tissues showed negligible expression of IP-10 message, with the exception of the spleen. Endotoxemic dogs demonstrated a robust induction of IP-10 mRNA in the heart, lung, kidney, liver, and spleen. Immunohistochemical studies indicated that IP-10 was predominantly localized in cardiac venular endothelial cells, bronchial epithelial cells, renal mesangial cells, and in the splenic red pulp of endotoxemic dogs. In addition, IP-10 expression was associated with T-lymphocyte infiltration in canine tissues. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) induced a marked upregulation of IP-10 message in canine venular endothelial cells. IP-10 expression in TNF-alpha-stimulated endothelial cells peaked at 6 h of stimulation and returned to baseline levels after 24 h. In addition, macrophage colony-stimulating factor (M-CSF) induced a dose-dependent induction of IP-10 mRNA in canine endothelial cells. M-CSF-mediated IP-10 expression peaked after 6 h of incubation and returned to baseline levels after 24 h. Canine endotoxemia is associated with a robust early expression of IP-10 in multiple tissues. IP-10 induction may be important in regulating lymphocyte recruitment and function. TNF-alpha, IL-1 beta, and M-CSF are potent inducers of IP-10 in canine endothelial cells and may indirectly mediate lymphocyte chemotaxis and activation in inflammatory processes.     

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.     

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

An abundant androgen-regulated mRNA in the mouse kidney.

We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.     

mPPP1R16B is a novel mouse protein phosphatase 1 targeting subunit whose mRNA is located in cell bodies and dendrites of neurons in four distinct regions of the brain

We cloned a cDNA encoding a novel mouse protein whose human homolog has been annotated in GenBank as a regulatory subunit of protein phosphatase 1, PPP1R16B. Both the primary protein sequence and the domain structure are highly conserved between PPP1R16B and proteins of unknown function from other species, such as Caenorhabditis elegans and Drosphila melanogaster. Besides a protein phosphatase 1 interaction motif, mouse PPP1R16B (mPPP1R16B) and the related proteins contain ankyrin repeats that may constitute binding sites for other proteins and C-terminal prenylation signals that are likely to target the proteins to the plasma membrane. In the adult mouse, Ppp1r16b mRNA is expressed in most tissues examined, with highest expression levels in kidney and brain. In the brain, Ppp1r16b message is particularly enriched in the olfactory bulb, striatum, dentate gyrus, and cerebellum. During postnatal cerebellar development, Ppp1r16b mRNA expression levels increase gradually and are maximal around postnatal day 30. In situ hybridization revealed that Ppp1r16b message is found in both the cell bodies and the dendrites in Purkinje cells of the cerebellum and granule neurons of the dentate gyrus.