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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 142 (1995), S. 139-150 
    ISSN: 1573-4919
    Keywords: canine idiopathic dilated cardiomyopathy ; calcium release channel ; mRNA ; oxidative phosphorylation ; myoglobin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Idiopathic dilated cardiomyopathy is associated with derangement of myocardial sarcoplasmic Ca-homeostasis and energy production. The molecular mechanism for these changes is unknown. Accordingly, we used genetic and experimentally-induced models of canine dilated cardiomyopathy and tested the hypothesis that these metabolic changes resulted from altered gene expression, as indicated by mRNA content. We studied dilated cardiomyopathy occurring naturally (n=9) in Doberman pinschers, and in dogs subjected to rapid ventricular pacing (n=5), in comparison with normal dogs (n=9). We determined content and integrity of mRNA's using Northern and slot blotting, and measured activities of their translated product for the Ca-release channel and Ca-ATPase of sarcoplasmic reticulum, lactate dehydrogenase of glycolysis, citrate synthase of the tricarboxylic acid cycle, and for myoglobin, ATP-synthetase and the adenine nucleotide transporter, which are integral in oxidative phosphorylation. We found that, whereas both mRNA content and enzyme activity for markers of Ca-cycling, glycolysis, and oxidative phosphorylation were downregulated (20–80%) in dilated cardiomyopathy, they were upregulated (10–15%) for tricarboxylic acid cycling and for ribosomal RNA. RNA from cardiomyopathic tissue was up to 50% more degraded than for normal hearts in association with a 150% increase in ribonuclease activity. Downregulation of the Ca-cycle was asymmetric, with the Ca-channel being 65% more affected than the Ca-ATPase. This work supports the general paradigm that transcriptional and translational responses to pathophysiology are major determinants of the metabolic response seen in cardiac failure.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 142 (1995), S. 131-138 
    ISSN: 1573-4919
    Keywords: cardiac muscle ; slow-twitch skeletal muscle ; sarcoplasmic reticulum ; Ca2+ uptake ; CaM kinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca2+-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca2+-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca2+-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca2+-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca2+-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 μM) directly to the Ca2+ transport assay medium results in minimal (∼ 112–130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition of either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca2+-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca2+ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca2+-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca2+-ATPase phosphorylation in the stimulation of Ca2+-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca2+ uptake and Ca2+-ATPase activity to phospholamban phosphorylation need to be re-examined.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 142 (1995), S. 151-161 
    ISSN: 1573-4919
    Keywords: hypocaloric feeding ; rat ; myocardium ; sarcoplasmic reticulum ; Ca-release channel ; Ca-ATPase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Hypocaloric feeding (HCF) depresses heart function causing cardiac atrophy, bradycardia, and decreased cardiac output. We tested the hypothesis that HCF results in decreased myocardial Ca- and ATP cycling. We reduced protein-calorie intake of adult rats by 20% for 7 days and then allowed them to recover for 3 days. Changes in ionized Ca concentration (nM/s) of 2.5% myocardial homogenates that were attributable to the Ca-ATPase pump and Ca-release channel (CRC), respectively, of the sarcoplasmic reticulum (SR) were depressed 41 and 85% by HCF from 61.6±9.4 and 24.7±3.3, to 36.1±2.8 and 3.6±2.9. Activity of the Ca-pump was restored after 3 days of refeeding, whereas the CRC remained 23% depressed (all p〈0.05). Additionally, the CRC activity was inhibited to a 3-fold greater extent than controls by HCF, but was disinhibited within one day of refeeding. The greater effect on CRC than Ca-pump activity resulted in net Ca-uptake being unaffected by HCF. In addition to depression of Ca-cycling, ATP sythetase and total ATPase activities (IU/g), respectively, were depressed 20 and 15% by HCF from 174±19 and 51.3±3.8 to 140±15 and 43.7±4.7, but were restored to control values within one day of refeeding. We conclude that HCF produces a compensatory, reversible, and asymmetric downregulation and inhibition of Ca-cycling, with the CRC being preferentially affected.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 145 (1995), S. 97-102 
    ISSN: 1573-4919
    Keywords: vanadium compounds ; osteoblast-like cells ; proliferation ; differentiation ; bone development ; growth factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of different vanadium compounds on proliferation and differentiation was examined in osteoblast-like UMR106 cells. Vanadate increased the cell growth in a biphasic manner, the higher doses inhibiting cell progression. Vanadyl stimulated cell proliferation in a dose-responsive manner. Similar to vanadate, pervanadate increased osteoblast-like cell proliferation in a biphasic manner but no inhibition of growth was observed. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity. These results suggest that vanadium derivatives behave like growth factors on osteoblast-like cells and are potential pharmacological tools in the control of cell growth.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The mitochondrial respiration rate and morphometric indices in endomyocardial biopsy samples were measured in 43 patients with dilated cardiomyopathy selected in accordance to WHO criteria by endomyocardial biopsy studies after excluding of various forms of myocarditis, alcoholic cardiomyopathy and other specific diseases of the heart. A group of 13 patients with unusually high mean myocyte diameter, 30±4 μm, and nuclear size, 57±5 μm, was selected. The remainder of patients (n=30) had significantly lower mean myocyte diameter and nuclear size, 23±3 and 42±6 μm, respectively, (p〈0.01). Creatine-stimulated elevation in mitochondrial respiration rate as measured in saponin-skinned was found in the former group to be much lower (36±4%) as compared with the remainder (90±12%). Also, the former group of patients had higher left ventricular enddiastolic pressure and volume index with concomitantly decreased ejection fraction. The results indicate that marked nuclear and cellular hypertrophy is associated with lower creatine-stimulated mitochondrial respiration rate and more severe cardiac failure. They suggest that disorders in energy supply to myofibrils may be related to disturbances in cellular genetic apparatus.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 143 (1995), S. 7-13 
    ISSN: 1573-4919
    Keywords: aestivation ; protein phosphorylation ; subcellular fractionation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of32P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 143 (1995), S. 15-20 
    ISSN: 1573-4919
    Keywords: aestivation ; protein synthesis ; stress protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Changes in [35S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 142 (1995), S. 171-178 
    ISSN: 1573-4919
    Keywords: interleukin-6 ; fibrinogen ; prostaglandin E2 ; interleukin-1 ; interleukin-1 receptor antagonist
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Infections, trauma and inflammatory processes induce a host response with increases in a large group of structurally and functionally diverse plasma proteins. Parental administration of foreign proteins also induce an increase in plasma fibrinogen. Interleukin-6 (IL-6) is a monocyte-derived mediator and has regulatory effects on acute phase protein genes which result in the induction of fibrinogen synthesis in primary hepatocytes, while the addition of interleukin-1 (IL-1) exerts a negative modulating influence on the IL-6-stimulated fibrinogen. In order to understand the mechanisms by which IL-1 inhibits IL-6-stimulated fibrinogen transcription and translation, and since IL-1 is believed to act through PGE2 stimulation, we have studied the influence of PGE2 in IL-6 or IL-1, alone and in combination, on Fg mRNA expression (by Northern blot analysis) and the influence of PGE2, indomethacin, and arachidonic acid on Fg secretion. Moreover, since human recombinant interleukin-1 receptor antagonist (hrIL-1ra) is a strong inhibitor of IL-1 induced IL-1 transcription and translation and has an inhibitory effect on PGE2, we have studied the effects of IL-1ra on the down-regulation of IL-6 stimulated fibrinogen by IL-1, using an Fg ELISA method. Herein we show that using human Hep 3B hepatoma cell cultures, steady-state levels of Fg mRNA is strongly increased in cells treated with IL-6 (10 ng/ml) and IL-6 (10 ng/ml)+PGE2 (5×10−6 M) compared to the control (Nil), while when IL-1 (1 ng/ml) was given in combination with IL-6 10 ng/ml, a strong inhibitory effect was found compared to IL-6 alone. The results were not statistically different from the controls. The addition of PGE2 at 5×10−6 M plus IL-6 10 ng/ml to the cell cultures induced an augmentation of Fg mRNA compared to IL-6 alone. Hep 3B hepatoma cells treated with IL-6, but not IL-1, induced an increase of Fg secretion compared to the control, while cells treated with the combination IL-6+IL-1 produced the same amounts of Fg compared to the control. The addition of PGE2 to IL-6 alone or IL-1+IL-6 did not modify the results. Arachidonic acid also had no effect on the combination IL-1 + IL-6. Similar results were obtained when indomethacin (5–50 μM) was used. When human recombinant IL-1 receptor antagonist (hrIL-1ra) 1–50 μg/ml was added to the Hep 3B hepatoma cell cultures, a dose-response restoration occured on IL-1-inhibited IL-6-stimulated Fg secretion. In these studies we show for the first time that PGE2 is not involved in the inhibition induced by IL-1 on IL-6 gene expression and secretion and that PGE2 (5×10−6 and 5×10−7 M) enhances IL-6-stimulation of Fg expression. Moreover, hrIL-1ra restored the down-regulation produced by IL-1 on IL-6-stimulated Fg translation. These results provide additional biological activities for IL-1, suggesting new and different mechanism(s) of action.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 143 (1995), S. 21-34 
    ISSN: 1573-4919
    Keywords: ribosomal proteins ; thermophilic eukaryotes ; electrophoresis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungusThermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved fromTh. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800–36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000–48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH4Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 155 (1996), S. 23-29 
    ISSN: 1573-4919
    Keywords: hexokinase type III ; pGEX-2T vector ; fusion protein ; glutathione S-transferase ; D-glucose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.
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