Different copy numbers of apparently identically deleted mitochondrial DNA in tissues from a patient with Kearns-Sayre syndrome detected by PCR

An apparently identical deletion of 4.977 bp in length (position 8,483-13,459) was detectable in the mitochondrial DNA from skeletal muscle, heart muscle, kidney, and liver of a patient with Kearns-Sayre syndrome. The proportion of deleted genome varied from 60% for the skeletal muscle to 15% for heart muscle and kidney, and was below 5% in the liver. The mtDNA heteroplasmy of the liver was only detectable after amplification by PCR. In skeletal and heart muscle histochemical and immunocytochemical findings concerning cytochrome c oxidase were in good correlation with the proportion of deleted mitochondrial DNA.     

Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.     

Bacterial infection and tissue-specific Hsp72, -73 and -90 expression in western painted turtles

Heat shock proteins (Hsps) are molecular chaperones that assist intracellular folding, assembly and translocation of proteins in prokaryotic and eukaryotic cells. A variety of stresses including hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation have been shown to increase the expression of Hsps. Likewise, bacterial infection represents a stress for the host cell. In this study, expression of the constitutive (Hsp73) and inducible (Hsp72) isoforms of Hsp70 and Hsp90 was monitored in brain, heart, liver and skeletal muscle from the western painted turtle Chrysemys picta bellii diagnosed with Septicemic Cutaneous Ulcerative Dermatitis (SCUD). This disease is caused by a gram-negative bacterium probably belonging to the Citrobacter spp. The expression of Hsp73 increased 1.8-fold in brain and liver, 2.2-fold in heart but did not change in skeletal muscle; Hsp72 expression increased 5.5-fold in brain and 3-fold in liver but did not change in heart or skeletal muscle; Hsp90 expression increased 9-fold in brain, 2.7-fold in heart and 2.4-fold in skeletal muscle but did not change in liver. These results suggest a tissue-specific Hsp response during bacterial infection and a role for Hsps in immunopathological events in reptiles.     

Effect of short- and long-term diabetes on carnitine and myo-inositol in rats.

1. The effect of short- (2 wk) and long-term (20 wk) streptozotocin diabetes was studied on urine, blood, liver, heart, brain, skeletal muscle, pancreas and kidney concentrations of acid-soluble carnitine and free myo-inositol. 2. Short-term diabetic rats excreted significantly higher concentrations of carnitine as well as myoinositol than normal rats. Blood carnitine and myo-inositol were not different between normal and diabetic rats. Diabetes caused a decrease in liver, brain and pancreatic carnitine, but not in heart, skeletal muscle and kidney. Myo-inositol concentration was decreased in liver, heart and kidney but not in brain, pancreas and skeletal muscle. 3. Long-term diabetic rats had higher urinary excretions of both carnitine and myo-inositol. Blood carnitine did not change; however, myo-inositol was higher in diabetic than in normal rats. Diabetes caused a significant increase in liver and a decrease in heart, brain, skeletal muscle and pancreatic content of carnitine; no difference in kidney carnitine was noted. Myo-inositol content was elevated only in liver of diabetic rats. 4. We suggest that carnitine and myo-inositol concentrations are influenced both by short- and long-term diabetes through changes in tissue metabolism.     

Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.     

Serum and organ creatine phosphokinase alterations in exercise.

Rats that swam for 3 h showed a 6-fold increase in serum creatine phosphokinase (SCPK) activity which declined to control values within 7 h after swimming. Of the excess SCPK, 77% was BB isoenzyme; the remainder was mainly MM with traces of MB. Kidney, liver, brain and lung contain mainly BB (50-80%) and only a trace of MB (0-7%). Heart CPK was composed of little BB (8%) and more MB (28%) and MM (64%). Skeletal muscle CPK was almost entirely MM. CPK activity is highest in skeletal muscle, intermediate in heart and brain and lowest in kidney, liver and lung. It is suggested that skeletal muscle and heart are not involved in CPK release in swimming, and kidney, liver and brain may be sites of release.     

耐力训练和急性力竭运行对大鼠组织金属硫蛋白含量的不同…

为探讨金属硫蛋白（ＭＴ）在运动提高机体自我贩作用,本文实验观察了游泳运动对大鼠心、肝、肺、脑、血管、因浆和骨骼肌等组织金属硫蛋白含量的影响。结果表明耐力训练组大鼠心、肝、肺和骨骼组织金属硫蛋白含量较政党对照组明显降低１３－３４％（Ｐ〈０．０５）;急性力竭运动组大鼠心、肝、脑、肺和骨骼肌组织其含量较正常对照则明显或高２１－７５％（Ｐ〈０．０５）;但两组大鼠血管和血浆ＭＴ含量变化与对照组大鼠要比无统计     

Effects of starvation on the tissular lipogenic rate in the obese Zucker rat.

The lipogenic rate of the obese rats was significantly higher than that of the lean rats in liver, white adipose tissue, skeletal muscle, heart and carcass. In the lean rats, a 24 h starvation period caused a significant decrease in the lipogenic rate of white adipose tissue and skeletal muscle while it increased that of heart, brain and brown adipose tissue. In the obese rats, starvation decreased the lipogenic rate in liver, skeletal muscle, white adipose tissue, brown adipose tissue and carcass. In spite of this, liver and skeletal muscle showed higher rates of lipid synthesis than the corresponding fed lean. It is concluded that starvation induces a qualitatively similar response in the obese versus the lean rat although the total lipogenic capacity of the animal is still higher.     

耐力训练和急性力竭运动对大鼠组织金属硫蛋白含量的不同影响

为探讨金属硫蛋白（ＭＴ）在运动提高机体自我保护能力方面的作用,本实验观察了游泳运动对大鼠心、肝、肺、脑、血管、血浆和骨骼肌等７种组织金属硫蛋白含量的影响。结果表明耐力训练组大鼠心、肝、肺和骨骼肌组织金属硫蛋白含量较正常对照组明显降低１３－３４％（Ｐ＜０．０５）;急性力竭运动组大鼠心、肝、脑、肺和骨骼肌组织其含量较正常对照组则明显升高２１－７５％（Ｐ＜０．０５）;但两组大鼠血管和血浆ＭＴ含量变化与对照组大鼠相比无统计学意义（Ｐ＜０．０５）。推测各组织金属硫蛋白在不同运动形式下的不同变化可能在运动提高机体自我保护能力方面具有积极意义。     

A study of iodothyronine 5'-monodeiodinase activities in normal and pathological tissues in man and their comparison with activities in rat tissues

The present study was undertaken to investigate the peripheral iodothyronine 5'-monodeiodination in different human and rat tissues. We studied iodothyronine 5'-monodeiodinase type I (5'-DI) activity in liver, kidney, intestine, right cardiac atrium and skeletal muscle and we compared the results with those in rat tissues. Lodothyronine 5'- monodeiodinase type II (5'-DII) activity was studied in normal and ischemic human heart and in rat normal myocardium and brain. The 5'-DI activity (fmol/min x mg protein) in liver and kidney was significantly higher (p < 0.001, ANOVA) in normal rat tissue than in human. However, no significant differences were observed in 5'-DI activity between normal and tumoral human intestine or between intestinal tissue of man and rat. 5'-DI activity in normal human skeletal muscle was significantly higher than that in rat skeletal muscle (p < 0.05). The 5'-DI activity was lower in human ischemic myocardium when compared to normal myocardium either in humans (p < 0.05) or rat (p < 0.001). The Km of 5'-DI was significantly lower in rat than in human kidney and liver (p < 0.05). We conclude that 1) 5'-DI is distributed widely among extrathyroidal human and rat tissues and 5'-DII activity is detectable both in human and rat heart; 2) 5'-DI activity in liver and kidney is lower in man than in rat; 3) 5'-DI activity in the skeletal muscle is higher in man than in the rat; 4) 5'-DI activity is decreased in tumoral tissues of human liver and kidney and in ischemic myocardium, while no significant difference was found between human and rat cardiac 5'-DII activity.     

Isolation and characterization of cDNAs encoding PDE5A,a human cGMP-binding,cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase

Human cGMP-binding, cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100 012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5′-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25–27.     

Effect of tamponade on the nucleotide composition and cytosine methylation level of DNA in the heart muscle of dogs

Base composition and quantitative changes in the 5-methylcytosine (m5c) content in DNA isolated from male dogs' heart muscle under the acute tamponade have been studied. Due to the effect of tamponade statistically reliable decrease (15%) in m5c content in DNA has been found to take place in experiment in comparison with control. Possible mechanisms of DNA methylation decrease by cytosine are under discussion.     

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.     

Measurement of beta-guanidinopropionate and phosphorylated beta-guanidinopropionate in tissues.

The Sakaguchi color reaction for monosubstituted guanidino compounds was applied to the measurement of β-guanidinopropionate and phosphorylated β-guanidinopropionate. The phosphorylated derivative was measured as an increase in β-guanidinopropionate following incubation with 0.1n HCl in a boiling-water bath for 10 min. After feeding rats 1% of β-guanidinopropionic acid in their diet for 69 days, skeletal muscle, heart, liver, kidney, and spleen contained 5–10 μmoles of a monosubstituted guanidino compound per gram wet weight of tissue. No β-guanidinopropionate was detected in brain or testes. Phosphorylated β-guanidinopropionate was found only in skeletal muscle (27 μmoles/g) and in heart (7 μmoles/g). Creatine hydrate (2%) added to the diet containing β-guanidinopropionic acid inhibited the accumulation of phosphorylated β-guanidinopropionate in the heart and partially inhibited its accumulation in skeletal muscle.     

Pyridoxamine (pyridoxine) phosphate oxidase activity in mammalian tissues

1. Vitamin B6-sufficient rats had moderate pyridoxamine-P oxidase specific activities in heart, brain, kidney and liver, but no detectable activity in skeletal muscle. Vitamin B6-deficiency in rats resulted in a decreased oxidase activity in liver but no change in the activities in other tissues. 2. The pyridoxamine-P oxidase activity in vitamin B6-sufficient mice was high in liver, moderate in brain and kidney, and not measurable in skeletal muscle and heart. Vitamin B6-deficient, compared with control mice, had decreased oxidase activities in brain, kidney and liver. 3. Mouse erythrocytes took up pyridoxine more rapidly than did rat and human erythrocytes. 4. Mouse and human erythrocytes rapidly converted pyridoxine to pyridoxal-P. Rat, hamster and rabbit erythrocytes had appreciably lower pyridoxamine-P oxidase activity than did mouse and human erythrocytes.     

Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)