The Helsinki Heart Study: an 8.5-year safety and mortality follow-up

MT-PVLT-10 transgenic mice express the large T-antigen of polyomavirus under the control of the mouse metallothionein-1 promoter. The males of this transgenic line developed testicular tumor and seminal vesicle engorgement at advanced ages. A novel partial cDNA was identified which hybridized to a 2.6 kilobase mRNA. The expression of this mRNA increased approximately two- to fifteen-fold in immortalized cell lines derived from testicular tumors as compared to similar cell lines derived from pre-adenomatous testes. The in vivo pattern of expression for this cDNA as well as its expression in various primary cultures and established cell lines derived from testis of MT-PVLT-10 mice is presented. Overlapping cDNA clones from liver, testes, and brain cDNA libraries containing the entire coding region for this novel cDNA have been isolated and sequenced. The coding region of this gene comprises 1179 nucleotides and predicts a polypeptide of 393 amino acids (calculated molecular mass 44,318). Motif analysis of the amino acid sequence has revealed that it contains several hydrophobic alpha-helices characteristic of transmembrane proteins.     

Molecular cloning and functional expression of feline thrombopoietin

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.     

Molecular analysis of the third component of canine complement (C3) and identification of the mutation responsible for hereditary canine C3 deficiency

Genetically determined deficiency of the third component of complement (C3) in the dog is characterized by a predisposition to recurrent bacterial infections and to type 1 membranoproliferative glomerulonephritis. The current studies were undertaken to characterize the cDNA for wild-type canine C3 and identify the molecular basis for hereditary canine C3 deficiency. Amplification, cloning, and sequence analysis indicated that canine C3 is highly conserved in comparison with human, mouse, and guinea pig C3. Southern blot analysis failed to show any gross deletions or rearrangements of DNA from C3-deficient animals. Northern blot analysis indicated that the livers of these animals contain markedly reduced quantities of a normal length C3 mRNA. The full-length 5.1-kb canine C3 cDNA was amplified in overlapping PCR fragments. Sequence analysis of these fragments has shown a deletion of a cytosine at position 2136 (codon 712), leading to a frameshift that generates a stop codon 11 amino acids downstream. The deletion has been confirmed in genomic DNA, and its inheritance has been demonstrated by allele-specific oligonucleotide hybridization.     

Molecular cloning and regulation of a novel guinea pig cytochrome P450 (CYP3A20) which differs from guinea pig CYP3A14 in only two amino acid residues

A full length cDNA clone of cytochrome P450, encoding 503 amino acid residues, was isolated from a male guinea pig liver cDNA library. The sequence was highly homologous to other members in the CYP3A subfamily and was designated CYP3A20. CYP3A20 and CYP3A14, another guinea pig CYP3A, shared 99.4% nucleotide and 99.6% deduced amino acid sequence homology. There were only two amino acid differences between CYP3A20 and CYP3A14. No significant induction of CYP3A20 mRNA in the livers from male guinea pigs treated with dexamethasone was observed by S1 mapping analysis although CYP3A14 mRNA was induced. The expression of CYP3A20 mRNA in the livers did not change between 5 and 10 weeks after birth while that of CYP3A14 mRNA in livers significantly increased at 10 weeks. This is the first report that the two highly resembling forms of cytochrome P450 display differently regulated expression from each other.     

A quantitative assessment of serum chylomicron by light scattering intensity: application to the intestinal fat absorption test

We cloned the canine interleukin-12 (IL-12) subunit cDNA. Canine IL-12 exhibited sequence homology to the known sequences of human, mouse, and bovine genes at nucleotide and amino acid levels. Cotransfection of the p35 and p40 subunits of canine IL-12 cDNA clones into COS-1 cells resulted in the secretion of IL-12, which supported proliferation of the stimulated canine lymphocytes, promoted induction of canine interferon-gamma (IFN-gamma) from canine lymphocytes, and showed antitumor effect in vitro. The cloned canine IL-12 will be useful for canine therapeutic applications.     

Kinetic analysis of two closely related receptor-like protein-tyrosine-phosphatases, PTP alpha and PTP epsilon

BACKGROUND: Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion. METHODS AND RESULTS: The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-alpha induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 microns in diameter. CONCLUSIONS: MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.     

Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro fertilization

Atlantic cod (Gadus morhua) transferrin cDNAs were isolated from a liver cDNA library using a cod transferrin-derived polymerase chain reaction product as a hybridization probe. The composite nucleotide sequence of two overlapping clones was 2223 bp in length excluding the poly(A) sequence and was equivalent to 87% of the 3' end of the Atlantic salmon transferrin cDNA sequence. Comparison of the deduced amino acid sequence of cod, salmon, Xenopus and several mammalian transferrins revealed that the two fish sequences are more similar with respect to their amino acid sequence and the position of additions/deletions than to other vertebrate transferrins. Conservation of the iron-binding domains and cysteine residues involved in disulphide bridges indicates that all transferrins share similar tertiary structure and support the hypothesis that extant vertebrate transferrin genes were derived from a gene duplication before the divergence of fish, frogs and mammals. Cod transferrin mRNA was detected in both brain and liver RNA and to a much lesser extent in RNA isolated from kidney and heart in contrast to salmon and several other vertebrates in which the transferrin gene is not expressed in brain.     

cDNA sequence analysis of the human brain insulin receptor

Brain tissue mRNA was amplified using polymerase chain reaction (PCR) with eight overlapping sets of primers that span the cDNA coding sequence for the human placental insulin receptor. Only the A isoform (lacking exon 11) of the receptor was detected. No difference was found in the predicted amino acid sequence of brain derived insulin receptor cDNA compared with the receptor from human placenta. A silent polymorphism was detected at nucleotide position 1698 (amino acid 523), confirming that mRNA corresponding to both alleles of the human brain receptor was sequenced. Our findings indicate that the unique glycosylation properties of brain insulin receptors do not stem from differences in primary structure, but rather are due to tissue-specific differences in post-translational processing.     

Identification and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana

Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix. A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana. The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species. Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side. Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods.     

Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney

We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of a cadmium-induced metallothionein from the insect Orchesella cincta (Collembola)

The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd-binding, cysteine-rich peptides were isolated from whole-body homogenates, using gel filtration and reversed-phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989-Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139-Da protein was unsuccessful, probably due to N-terminal blockage. Using different PCR techniques (3' and 5' RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C-terminal part of this protein. The 4139-Da protein is probably encoded by the N-terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post-translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.     

Molecular cloning of a bovine ornithine decarboxylase cDNA and its use in the detection of restriction fragment length polymorphisms in Holsteins

A cDNA coding for ornithine decarboxylase (ODC) was isolated from a bovine liver cDNA library. The clone (1758 base pairs) consisted of 5'- and 3'-untranslated regions of 185 and 187 nucleotides, respectively, and an open reading frame of 1383 nucleotides encoding an ODC protein (M(r) 51,342 daltons) of 461 amino acids. Comparison of the nucleotide and the predicted amino acid of the cDNA with other mammalian ODCs showed a very high degree of homology both at the DNA and protein levels. The bovine ODC mRNA was identified by northern blot to be a single species with a molecular size of 2.35 kilobase pairs. Primer extension analysis indicated that the 5'-untranslated region of the bovine ODC mRNA was 312 nucleotides long. Southern blot analysis of bovine genomic DNA revealed restriction fragment length polymorphisms when cleaved with restriction enzymes PstI, MspI, TaqI, and Bg/I.     

Determinants of cholesterol screening and treatment patterns. Insights for decision-makers

We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.     

Identification and characterization of a novel human aldose reductase-like gene

We have identified a novel human protein that is highly homologous to aldose reductase (AR). This protein, which we called ARL-1, consists of 316 amino acids, the same size as AR, and its amino acid sequence is 71% identical to that of AR. It is more closely related to the AR-like proteins such as mouse vas deferens protein, fibroblast growth factor-regulated protein, and Chinese hamster ovary reductase, with 81, 82, and 83%, respectively, of its amino acid sequence identical to the amino acid sequence of these proteins. The cDNA of ARL-1 was expressed in Escherichia coli to obtain recombinant protein for characterization of its enzymatic activities. For comparison, the cDNA of human AR was also expressed in E. coli and analyzed in parallel. These two enzymes differ in their pH optima and salt requirement, but they act on a similar spectrum of substrates. Similar to AR, ARL-1 can efficiently reduce aliphatic and aromatic aldehydes, and it is less active on hexoses. While AR mRNA is found in most tissues studied, ARL-1 is primarily expressed in the small intestines and in the colon, with a low level of its mRNA in the liver. The ability of ARL-1 to reduce various aldehydes and the locations of expression of this gene suggest that it may be responsible for detoxification of reactive aldehydes in the digested food before the nutrients are passed on to other organs. Interestingly, ARL-1 and AR are overexpressed in some liver cancers, but it is not clear if they contribute to the pathogenesis of this disease.     

Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.