Protein kinase Cδ promotes proliferation and induces malignant transformation in skeletal muscle

In this paper, we investigated the isoform‐specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase‐negative mutant of nPKCδ (DN‐nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN‐nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, β, ε) exerted only minor (mostly growth‐inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth‐promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Functional and adaptive significance of differentially expressed lactate dehydrogenase isoenzymes in tissues of four obligatory air-breathing Channa species

This study investigates the differential expression of lactate dehydrogenase (LDH) isoenzymes in the genus Channa using PAGE. With the help of obligate air-breathing, all of the selected species can sustain water deprivation to varying degrees. In subunit composition and higher electrophoretic mobility of LDH-A₄, the profiles of channid species were similar to other teleosts documented in the literature. However, inter- and intra-species differences, with particular reference to aerobic/anaerobic metabolic options, existed. Whereas glycolysis in Channa punctata appears to depend largely on aerobic LDH-B and partly on anaerobic LDH-A, metabolism in C. gachua, C. striata and C. marulius depends exclusively on the activity of anaerobic LDH-A. Expression of the third locus Ldh-C was recorded in the eyes of C. marulius, in addition to C. gachua. Heat inactivation experiments reveal species differences between LDH isoenzymes and a general order of the relative stabilities: LDH-C > DH-B > LDH-A. Metabolic and evolutionary implications of the findings have also been discussed.     

Protective effects of antioxidants on age-related changes in the electrophoretic patterns of cardiac LDH,hepatic ALP and serum proteins in male golden hamster

Basal and antioxidant-induced changes in the isoenzyme and isoform patterns of cardiac lactate dehydrogenase (EC 1.1.1.27) and hepatic alkaline phosphatase (EC 3.1.3.1), respectively, as well as the electrophoretic patterns of serum proteins in different age groups of male golden hamster were compared. This is to test whether age-induced changes could be corrected by long-term administration of antioxidants. Data indicated that aging causes no remarkable change in the total activity of either cardiac LDH or hepatic ALP, however a significant increase in the fractional activity of some cardiac LDH isoenzymes and a significant reduction in the fractional activity of some hepatic ALP isoforms were induced by aging. On the other hand, long-term administration of antioxidants appeared to manifest a clear counteracting effect on the age-related changes in old age. This effect was indicated in the fractional activity of cardiac LDH isoenzymes and of hepatic ALP isoforms. The present study has also shown a wide-range variation in serum protein patterns due to aging and/or antioxidant administration, which indirectly reflect a parallel variation in the process of gene expression and/or proteolytic activity.     

"Chitin-specific" peroxidases in plants

The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega (Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.     

Isoenzyme differentiation of Aedes aegypti populations in French Guiana

Population genetics of peri-domestic Aedes aegypti (Diptera: Culicidae), vector of dengue and yellow fever, were investigated by gel electrophoresis of 10 enzyme loci in 14 samples of mosquito larvae collected in 1997-1998 from localities separated by distances of 3-275 km in French Guiana. Genetic differentiation between geographical populations was generally high (mean FST = +0.111, P < 10(-5)) even among seven sites <30 km apart (FST = +0.137, P < 0.05), but not positively correlated with distance. Thus, Ae. aegypti comprises a mosaic of genetically differentiated populations in French Guiana. This may be attributed to reinvasion from diverse origins through repeated founder events after this vector species was eliminated during the 1940s to 1960s.     

Purification of Cu, Zn-Superoxide Dismutase Isoenzymes from Fish Liver: Appearance of New Isoforms as a Consequence of Pollution

Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu, Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize their utility as biomarkers, Cu, Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H₂O₂ concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. The pure isoenzyme V generated only a new band of pI 5.0. Concomitant with oxidation, the activity of peaks I and V was lost in a H₂O₂ concentration-dependent manner. The pattern of the new acidic bands generated upon the oxidixing treatment of isoenzyme I closely resembles that observed in crude extracts from polluted animals.     

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

ABSTRACT. Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi , demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.