Rat transthyretin (prealbumin). Molecular cloning, nucleotide sequence, and gene expression in liver and brain

Transthyretin cDNA was isolated from a rat liver cDNA library. Analysis of the nucleotide sequence revealed a signal peptide-like sequence preceding a section coding for a full length subunit and an untranslated sequence at the 3' end. The deduced primary structure of rat transthyretin was compared with that of human transthyretin. It was highly conserved at the binding sites for thyroxine and the interfaces and core regions of the subunits. The cDNA for transthyretin was used to measure mRNA levels by hybridization. During acute inflammation, the amount of transthyretin mRNA in liver decreased (reaching a minimum of 25% of the normal level 36 h after inducing inflammation), suggesting regulation of transthyretin synthesis at the mRNA level. Transthyretin mRNA was found only in the liver and in the choroid plexus, but not in other parts of the central nervous system nor in the adrenal glands, kidney, spleen, testes, heart, lung, intestine, and ovaries. One gram of choroid plexus contained about 25 times larger amounts of transthyretin mRNA than 1 g of liver. By synthesizing an important hormone carrier protein, the choroid plexus may be an important link in the chemical communication between the central nervous system and the bloodstream.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

Induction of rat liver angiotensinogen mRNA following acute inflammation

Inflammatory responses of the angiotensinogen mRNA in rat liver and brain were examined by RNA blot-hybridization analysis with use of a cDNA probe specific for rat angiotensinogen. The angiotensinogen mRNA in the liver increased rapidly during the first 5 h following the administration of Escherichia coli lipopolysaccharide, and at maximum level of induction, the mRNA increased approximately 5-fold over its normal level. The levels of the mRNA increased with increasing doses of lipopolysaccharide, the half-maximal dose being approximately 1 microgram/100 g body weight. In contrast, no such increase was observed in the brain angiotensinogen mRNA. Thus, the expression of the rat angiotensinogen mRNA is regulated in a tissue-specific manner in response to induction of acute inflammation.     

Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.     

Cloning and expression analysis of the sheep ceruloplasmin cDNA.

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.     

Expression of ceruloplasmin gene in mammalian organs from the data of hybridization analysis with complementary DNA probes

Expression of ceruloplasmin (Cp)-coding gene in rat and human liver and brain tissues was studied by Northern blot hybridization and by in situ hybridization with cloned species-specific cDNA probes. In rat brain structures, different levels of Cp mRNA were detected, the maximal one was found in cerebellum. The steady-state level of Cp mRNA in rat and human brain was several times lower than in parenchymatous liver cells. The size heterogeneity of Cp mRNA was found. Polyadenylated RNA prepared from human liver contains two equally abundant Cp mRNAs differing in their chain length (3.6 and 4.5 kb) while brain polyadenylated RNA contains a single Cp mRNA (4.5 kb).     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Major acute-phase alpha(1)protein in the rat: structure, molecular cloning, and regulation of mRNA levels

Rat major acute-phase alpha(1)protein (MAP) was characterized by determining its secondary structure, ligand binding and partial amino acid sequence. A cDNA clone expressing MAP and coding for the entire mature protein was isolated from a cDNA library in E. coli prepared from rat liver mRNA. By hybridization to nick translated cDNA, mRNA for MAP was found only in liver, where it increased 17-fold during acute inflammation. Constant proportions of rates of leucine incorporation into MAP over mRNA levels in liver indicated that the regulation of the synthesis of MAP is due to a change in the rate of synthesis and/or the stability of mRNA for MAP, but not the rate of its translation.     

IL-6 functions as an exocrine hormone in inflammation. Hepatocytes undergoing acute phase responses require exogenous IL-6

We have previously shown that IL-6 is the major monocyte- and fibroblast-derived regulator of acute phase protein gene expression and synthesis in hepatocytes in inflammation. Recently, we and others have shown that rat and human hepatoma cells express IL-6 mRNA, and the question arose as to whether normal hepatocytes express IL-6 and whether any such expression occurs under normal physiologic conditions or is seen in inflammation. Poly A+ mRNA of liver from normal rats and from rats undergoing an acute phase response was not positive when probed with cDNA for rat IL-6 under conditions in which macrophage mRNA was strongly positive. We then compared poly A+ mRNA from purified hepatocytes freshly isolated from normal rats--from rats that were undergoing an acute inflammatory response and from freshly isolated normal hepatocytes that had been cultured for 24 h in the presence or absence of dexamethasone (microM). Only the mRNA from normal hepatocytes cultured for 24 h in the absence of any glucocorticoid was obviously positive for IL-6. The increased expression of gamma-fibrinogen mRNA indicated the presence of inflammation. These results confirm the identification of IL-6 as an exogenous hormone for regulating normal hepatic acute phase protein synthesis in inflammation and rules out an autocrine mechanism being active in the liver in normal homeostasis.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats

Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.     

Molecular cloning of cDNA for rat and human carbamyl phosphate synthetase I

Recombinant plasmids with inserts complementary to the mRNA for carbamyl phosphate synthetase I were identified from a rat liver cDNA library by hybrid-selected mRNA translation. Four clones, the largest being 3100 base pairs, were identified for the rat liver enzyme. Using the rat liver cDNA as a probe, two homologous recombinant plasmids of approximately 1200 base pairs in length were isolated from a human liver cDNA library. Northern blot analysis of rat liver mRNA and baboon liver mRNA revealed the presence of a 5000-base mRNA homologous to both rat and human cDNA probes. No homologous mRNA was observed in mRNA from rat heart or rat kidney as is consistent with the known tissue distribution of this enzyme. The induction of carbamyl phosphate synthetase and argininosuccinate synthetase mRNA during the fetal and postnatal development of the rat was studied by dot blot analysis of isolated mRNA. The mRNA for both enzymes appeared between 17 and 19 days of fetal life and reached approximately 40% of adult levels during this period. This initial increase was followed by a rapid decline just prior to birth. The mRNA levels slowly increased during postnatal life, not reaching adult levels until after the 20th day of neonatal life. Using the human cDNA clones, the human carbamyl phosphate synthetase gene was mapped to chromosome 2 utilizing a panel of Chinese hamster X human somatic cell hybrids. Analysis of one hybrid with a human-Chinese hamster translocation provided a provisional assignment to the short arm of chromosome 2.     

Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross-hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar. Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5-fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of D-galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.