ABSTRACT
In the 1950s to 1970s developed countries reported declines in populations of raptorial and fish-eating birds and dichlorodiphenyltrichloroethane (DDT) and its metabolites were considered causative substances because they accumulated significantly in the tissues of wild birds and animals. However, except for the estrogenic effects of o,p'-DDT, a minor component of commercial DDT, there has been no compelling evidence that DDT directly affects avian reproductive systems. To assess the possible impact of DDT on development and reproduction of birds, exposure experiments to the major component of commercial DDT, p,p'-DDT, and its persistent metabolite, p,p'-dichlorodiphenyldichloroethylene (DDE), were performed using Japanese quail (Coturnix japonica) eggs; the test substances (3 to 100 µg/g) were injected into the yolk prior to incubation, and hatched chicks were raised to adulthood. p,p'-DDT had no significant effects on the morphology and function of the reproductive systems, although the hatchability of treated eggs was reduced at the highest dose (100 µg/g). High doses of p,p'-DDE slightly enhanced the eggshell forming ability of female quails; eggshell mass and thickness were increased at 30 µg/g or more although no morphological changes were observed in the oviduct. Transcriptions of the CYP11A1 gene in the ovaries, and of AHR and ARNT in the livers, of adult females were significantly increased at 3 µg/g or more of p,p'-DDT. Except for low hatchability, transovarian exposure to p,p'-DDT or p,p'-DDE did not markedly impair the avian reproductive systems, but the hormonal actions of these compounds are likely to change reproductive and hepatic functions even after maturation.
Subject(s)
Coturnix/physiology , DDT/adverse effects , Dichlorodiphenyl Dichloroethylene/adverse effects , Embryo, Nonmammalian/drug effects , Ovum/drug effects , Reproduction/drug effects , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Cholesterol Side-Chain Cleavage Enzyme/genetics , Dose-Response Relationship, Drug , Embryo, Nonmammalian/physiology , Embryonic Development/drug effects , Female , Liver/metabolism , Ovary/enzymology , Oviducts/drug effects , Ovum/physiology , Receptors, Aryl Hydrocarbon/metabolism , Transcription, Genetic/drug effectsABSTRACT
It has been reported that green tea catechins enhance the force of contraction of isolated heart muscle preparations. However, it remains controversial whether or not the increase in force of contraction is related to an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i). In this study, the relationship was investigated using a left atrial muscle preparation isolated from guinea pig heart. In the left atrial muscle preparations without fura-2/AM loading, neither EGC (epigallocatechin) nor EC (epicatechin) influenced the force of contraction, but EGCG (epigallocatechin gallate) and ECG (epicatechin gallate) increased the force of contraction in a dose-dependent manner. The ED(50) value of EGCG was significantly higher than that of ECG. In the atrial muscle preparations loaded with fura-2/AM, EGCG and ECG increased the amplitude of [Ca(2+)]i(peak [Ca(2+)]i minus diastolic [Ca(2+)]i) which is associated with the increase in force of contraction. Simple regression analysis between the degree of increase in the force of contraction and the increase in the amplitude of [Ca(2+)]i revealed a positive correlation in EGCG, ECG and CaCl(2). In addition, the slopes of the regression lines of EGCG and ECG were comparable with those of CaCl(2). It was suggested that atrial muscle preparations had a higher affinity for ECG than EGCG, and that the increase in the force of contraction by EGCG and ECG was closely related to the increase in the amplitude of [Ca(2+)]i.
Subject(s)
Calcium/metabolism , Camellia sinensis/chemistry , Heart Atria/drug effects , Myocardial Contraction/drug effects , Plant Extracts/pharmacology , Animals , Catechin/analogs & derivatives , Catechin/chemistry , Catechin/pharmacology , Dose-Response Relationship, Drug , Guinea Pigs , Male , Molecular Structure , Regression AnalysisABSTRACT
A 7-year-old, male, mixed breed dog was referred to the Veterinary Teaching Hospital at Kitasato University because of anorexia, lameness and multiple cutaneous lesions. Observation of bone marrow plasmacytosis, osteolytic bone lesions, serum myeloma protein and cutaneous infiltration of myeloma cells led us to a diagnosis of multiple myeloma (MM) with cutaneous involvement. Polymerase chain reaction and sequence analysis for the rearranged genes of immunoglobulin and T-cell receptor demonstrated that the neoplastic cells found in skin lesions or bone marrow are of B-lymphocyte lineage and share a common original precursor cell. The dog was treated with UW-Madison protocol or melphalan/prednisone protocol and survived 175 days. This is rare case of anaplastic MM with cutaneous involvement in dog.
Subject(s)
Dog Diseases/pathology , Multiple Myeloma/veterinary , Skin Neoplasms/veterinary , Animals , Antineoplastic Combined Chemotherapy Protocols , Dog Diseases/drug therapy , Dogs , Fatal Outcome , Lomustine/therapeutic use , Male , Melphalan/administration & dosage , Melphalan/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Prednisolone/administration & dosage , Prednisolone/therapeutic use , Skin Neoplasms/drug therapy , Skin Neoplasms/pathologyABSTRACT
It has been proposed that the cardiotoxicity of anthracycline anticancer drugs involves free-radical formation. One early manifestation of toxicity appears to be caused by the antimuscarinic actions of these drugs. Accordingly, we examined whether the antimuscarinic action of one of these drugs, doxorubicin, is altered by antioxidants. In electrically stimulated left atrial muscle preparations obtained from guinea pig hearts, doxorubicin significantly increased the tissue concentration of thiobarbituric acid-reactive substance indicating increased lipid peroxidation. This effect of doxorubicin was significantly suppressed by the antioxidants alpha-tocopherol, dexrazoxane, and epigallocatechin gallate. Carbachol produced a concentration-dependent negative inotropic effect in our atrial preparations. Doxorubicin caused a seemingly parallel rightward shift of the concentration-response curve for carbachol. Neither alpha-tocopherol, dexrazoxane, nor epigallocatechin gallate reversed this effect of doxorubicin. The results indicate that in extirpated heart tissue, doxorubicin causes lipid peroxidation through the formation of free radicals. However, this effect of doxorubicin is unrelated to its antimuscarinic action.
Subject(s)
Doxorubicin/pharmacology , Free Radicals/metabolism , Heart/drug effects , Heart/physiology , Muscarinic Antagonists/pharmacology , Animals , Dose-Response Relationship, Drug , Guinea Pigs , Male , Myocardium/metabolismABSTRACT
We reported previously that doxorubicin, an anticancer agent that has an anthracycline structure, alters Ca2+ releasing and uptake mechanisms in the sarcoplasmic reticulum of myocardial cells. These effects of doxorubicin are apparently related to its cardiotoxicity. Mitoxantrone is a similar anticancer agent with an anthracenedion structure that has been shown to be significantly less cardiotoxic. In the present study, the effects of mitoxantrone on the functions of the sarcoplasmic reticulum were examined in isolated muscle preparations obtained from the guinea-pig heart. In electrically-stimulated left atrial muscle preparations, incubation in vitro for 4 hr with 30 or 100 microM mitoxantrone significantly prolonged the time to the peak of twitch tension, markedly increased the developed tension observed at lower stimulation frequencies, thereby attenuating the slope of positive force-frequency relationships, and increased the postrest contraction observed after a 60-sec quiescent period. In myocytes isolated from ventricular muscles, 30 microM mitoxantrone increased the peak and the size of intracellular Ca2+ concentrations ([Ca2+] i), and prolonged the time to peak [Ca2+]i. In skinned muscle fiber preparations obtained from the left ventricular muscle, 30 muM mitoxantrone significantly increased the caffeine-induced contraction without affecting the Ca2+ sensitivity of contractile proteins. These results suggest that mitoxantrone enhances Ca2+ release from the sarcoplasmic reticulum in isolated atrial muscle preparations obtained from the guinea-pig heart. Apparent enhancement of the sarcoplasmic reticulum functions, in contrast to anthracyclines that has been shown to suppress these functions, seems to explain the relative lack of marked cardiotoxicity of mitoxantrone.
Subject(s)
Antineoplastic Agents/toxicity , Cardiotoxins/toxicity , Doxorubicin/toxicity , Heart/drug effects , Mitoxantrone/toxicity , Myocardial Contraction/drug effects , Sarcoplasmic Reticulum/drug effects , Analysis of Variance , Animals , Antineoplastic Agents/metabolism , Calcium/metabolism , Cardiotoxins/metabolism , Doxorubicin/metabolism , Electric Stimulation , Fluorescence , Guinea Pigs , Male , Mitoxantrone/metabolismABSTRACT
Previous studies on human uterine and placental tissues have found variants, derived from alternatively spliced mRNAs, of preproendothelin-2 (PPET2) that lack a post-translational proteolytic site essential for normal processing. Here we report a splice variant of cat PPET2 mRNA expressed in the stomach. After cloning the full-length cDNA of cat PPET2, organ distribution analysis of the transcript by reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. In addition to the fragment with a size predicted based on the cDNA sequence obtained by cloning, an additional PCR fragment of smaller size was detected in stomach tissue. Subsequent cloning and sequence analysis of the smaller PCR product demonstrated that it derives from a splice variant with full-length deletion of a region corresponding to exon 4 of the PPET2 gene.
Subject(s)
Alternative Splicing/genetics , Cats/metabolism , Endothelins/metabolism , Protein Precursors/metabolism , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cats/genetics , Cloning, Molecular , DNA Primers , DNA, Complementary/genetics , Endothelins/genetics , Gastric Mucosa/metabolism , Molecular Sequence Data , Protein Precursors/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Angiotensin-converting enzyme (ACE; EC 3.4.15.1), a dipeptidyl carboxypeptidase, converts angiotensin I to angiotensin II, the central product of the renin-angiotensin system. We here report molecular cloning of the complete open reading frame (ORF) of hamster somatic-type ACE and its expression in hamster organs. The cloned cDNA comprises an ORF of 3942 bp, which encodes 1314 amino acids of the precursor protein of hamster somatic ACE. On the deduced amino acid sequence a putative signal peptide and a transmembrane segment are predicted at the N-terminus and near the C-terminus, respectively. Two homologous domains, referred to as N- and C-domains, are present within somatic ACE, and within each of the homologous domains a putative active center is found, as has been the case in human, mouse, rat and rabbit. The similarity of the hamster sequence with the sequences of these other mammals at both the nucleotide and amino acid levels is high (above 83%). mRNA expression analysis by conventional polymerase chain reaction (PCR) shows wide distribution of the transcript, with dominant expression in lung and kidney. Quantitative analysis of mRNA expression demonstrates that levels in lung and kidney are 100-1000 times higher than in the other organs, suggesting that these organs are important in the hamster renin-angiotensin system, as they are for other mammals.
Subject(s)
Cloning, Molecular , DNA, Complementary , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/isolation & purification , RNA, Messenger/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cricetinae , Molecular Sequence Data , Peptidyl-Dipeptidase A/biosynthesisABSTRACT
The full-length cDNA of dog preproendothelin-3 (PPET3) was cloned from lung tissue using RT-PCR and rapid amplification of cDNA ends. Aside from the poly (A) tail, the full-length cDNA was 1976 bp. A polyadenylation signal sequence and one copy of a consensus sequence, ATTTA, which is related to mRNA turnover, was found in the 3' noncoding region. The cDNA had a 594-bp open reading frame encoding a 198-amino acid polypeptide. Regions corresponding to a bioactive mature ET3 peptide, an intermediate form known as big-ET3, and an ET3-like peptide were observed in dog PPET3. Expression of PPET3 mRNA was detected throughout the organs examined, which included heart, lung, liver, kidney, spleen, stomach, pancreas, duodenum, colon, uterus, ovary and testis.
Subject(s)
Dogs/genetics , Endothelins/genetics , Protein Precursors/genetics , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , DNA, Complementary/genetics , Endothelins/metabolism , Molecular Sequence Data , Protein Precursors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
To compare the structure of the precursor polypeptide of dog endothelin-3, preproendothelin-3 (PPET-3), with the PPET-3 of other mammals, we cloned dog cDNA from lung tissue using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. An open reading frame encoding a 198-amino-acid polypeptide was found in the cDNA. Regions corresponding to a bioactive mature endothelin-3 peptide, an intermediate form known as big-endothelin-3 and an endothelin-3- like peptide were observed in the putative PPET-3. Comparative analysis showed that the similarity of the dog open reading frame sequence with those from human hypothalamus, mouse intestine, and rat eye is 76.2%, 69.5% and 66.3%, respectively, and that the similarity at the amino acid level is 65.6%, 59.8% and 58.8%, respectively. RT-PCR demonstrated significant elevated expression of PPET-3 mRNA in the kidney of dog with interstitial nephritis.
Subject(s)
Endothelin-3/chemistry , Kidney/chemistry , Nephritis, Interstitial/metabolism , Protein Precursors/chemistry , Amino Acid Sequence , Animals , Cloning, Molecular , Disease Models, Animal , Dogs , Endothelin-3/genetics , Humans , Mice , Molecular Sequence Data , Nephritis, Interstitial/genetics , Open Reading Frames , Protein Precursors/genetics , RNA, Messenger/analysis , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Up-RegulationABSTRACT
We cloned and characterized horse preproendothelin-2 (PPET-2) cDNA from intestinal tissue. The cDNA encoded 178 amino acids of the PPET-2 polypeptide, in which a 21-amino-acid mature endothelin-2 peptide and a 16-amino acid endothelin-2-like peptide were found. For the open reading frame the correspondence of horse PPET-2 cDNA with those of the ferret, human, dog, mouse and rat was 85.1%, 84.9%, 82.1%, 77.8% and 77.2%, respectively. Analysis of the organ distribution of PPET-2 mRNA by reverse transcription-polymerase chain reaction demonstrated that the kidney, stomach and small intestine are major sites of expression of the PPET-2 gene. Surprisingly, the mRNA is not detected in the large intestine, where high expression is demonstrated in the mouse and rat. This difference may result from the underlying functional differences of the large intestine between a herbivore (horse) and an omnivore (mouse and rat).
Subject(s)
Cloning, Molecular , Endothelin-2/genetics , Protein Precursors/genetics , Sequence Analysis, DNA , Sequence Analysis, Protein , Amino Acid Sequence , Animals , Base Sequence , Dogs , Endothelin-2/chemistry , Ferrets , Horses , Humans , Intestine, Small/chemistry , Kidney/chemistry , Mice , Molecular Sequence Data , Protein Precursors/chemistry , RNA, Messenger/analysis , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Stomach/chemistryABSTRACT
Endothelin-2 (ET2) is a member of the endothelin family of 21-amino acid peptides with vasoconstrictive activity. We report here the molecular cloning of the canine full-length cDNA of the precursor form of ET2, prepro-ET2 (PPET2), from intestinal tissue by means of reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'- and 3'-rapid amplification of cDNA ends (RACE). Aside from the poly (A) tail the cDNA was found to be 1195 bp and included an open reading frame of 534 bp encoding a PPET2 polypeptide of 178 residues, in which the regions corresponding to bioactive mature ET2 peptide, an intermediate form big-ET2, and endothelin-like peptide are found. The organ distributions of PPET2 mRNA and a splicing variant were analyzed by RT-PCR. PPET2 transcript was detected in duodenum, colon, stomach, lung, liver, uterus, ovary, testis and kidney, but not in spleen. A splicing variant was found in none of the organs. Thus, based on the cloned cDNA sequence, we established a quantitative assay for dog PPET2 mRNA level using a real-time PCR system. Quantitative analysis by this method in various organs of the dog demonstrated that the dominant gene expression occurs in the intestine, with higher expression in large intestine than in small intestine.
Subject(s)
Cloning, Molecular , Endothelins/genetics , Gene Expression Profiling , Protein Precursors/genetics , Sequence Homology, Nucleic Acid , Animals , Base Sequence , Chromosome Mapping , DNA Primers , DNA, Complementary/genetics , Dogs , Molecular Sequence Data , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Endothelin-2 (ET2), which was originally identified in human, is a bioactive peptide of 21 amino acids with strong vasoconstrictive and pressor effects. Here we report the cDNA cloning and characterization of bovine preproendothelin-2 (PPET2), the precursor form of ET2. The bovine cDNA encodes 177 amino acids of the PPET2 polypeptide, in which a 21-amino acid mature ET2 peptide and a 16-amino acid ET2-like peptide as well as a 23-amino acid putative signal peptide were found. The bovine ET2-like peptide sequence was missing a dibasic amino acid pair at the C-terminal, in contrast to human, mouse and rat, for which the ET2-like sequence is flanked by dibasic pairs at both the N- and C-terminals. Gene expression analysis by RT-PCR showed that the transcript is expressed in various organs including heart, lung, liver, kidney, gastrointestinal tract, uterus and ovary, but not in spleen. Within the gastrointestinal tract, gene expression was detected in rumen, a ruminant-specific digestive organ, as well as stomach, duodenum and colon.
Subject(s)
Cattle/genetics , Endothelins/genetics , Protein Precursors/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle/metabolism , Chromosome Mapping , DNA, Complementary/genetics , Gene Expression , Gene Expression Profiling , Molecular Sequence Data , Oligonucleotides/genetics , Organ Specificity , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.
Subject(s)
Endothelins/genetics , Intestinal Mucosa/metabolism , Lung/metabolism , Animals , Animals, Newborn , Endothelin-1/genetics , Endothelin-2/genetics , Female , Gestational Age , Intestines/embryology , Lung/embryology , Male , Mice , Mice, Inbred ICR , Polymerase Chain Reaction/methodsABSTRACT
Ferret preproendothelin-2 (PPET2) cDNA was cloned from intestinal tissue by reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'- and 3'-rapid amplification of cDNA ends (RACE). The cDNA comprises 1230 bp, excluding the poly(A) tail, and has 534 bp of open reading frame encoding a putative polypeptide of 178 residues, in which a 21-amino acid mature endothelin-2 (ET2) peptide as well as a 24-amino acid putative signal peptide and a 16-amino acid ET2-like peptide were found. The homology of the full-length cDNA sequence of ferret with those of horse, human, mouse, or rat was 75.6, 71.6, 65.4 or 65.1%, respectively, and the homology of the coding region was 85.1, 81.6, 78.1 or 75.3%, respectively. Phylogenetic analysis among ferret, horse, human, mouse, rat and dog showed that ferret has a closer relationship to dog than to the other mammals.