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PubMed2004

Bacterial mRNA purification by magnetic capture-hybridization method.

A magnetic capture-hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A(260 nm) / A(280 nm) value, denatured gel electrophoresis and RT-PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture-hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.

Bacteria↗
PubMed1992

Inhibition of rabies virus transcription in rat cortical neurons with the dissociative anesthetic ketamine.

In a previous study (B. P. Lockhart, H. Tsiang, P. E. Ceccaldi, and S. Guillemer, Antiviral Chem. Chemother. 2:9-15, 1991), we demonstrated an antiviral effect of the general anesthetic ketamine for rabies virus in neuronal cultures and in rat brain. This report describes an attempt to determine at what level ketamine acts on the rabies virus cycle in rat cortical neuron cultures. Immunofluorescence and [35S]methionine labelling of infected neurons showed that ketamine (1 to 1.5 mM) inhibited viral nucleoprotein and glycoprotein syntheses. Northern (RNA) blots of total RNA from drug-treated neurons, hybridized with 32P-labelled oligonucleotide probes for rabies virus nucleoprotein, matrix protein, and glycoprotein genes, showed a marked reduction (5- to 11-fold) in the levels of rabies virus mRNAs, relative to those in untreated neurons. No significant change in the levels of cellular beta-actin mRNA were detected in ketamine-treated cells. A similar antiviral effect was observed with MK-801; however, no inhibition of rabies virus synthesis was observed with the general anesthetic chloral hydrate. The antiviral effect was not complete; a time-dependent recovery of viral transcription and rabies virus protein synthesis was observed, but no infectious virus was released into the culture supernatant. The lack of any modification of cellular protein or mRNA synthesis by ketamine suggests an antiviral mechanism acting at the level of rabies virus genome transcription.

Animals↗
PubMed1996

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

Amino Acid Sequence↗
PubMed1999

Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues.

Precise and sensitive quantitation of viral RNA in specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals has become an indispensable tool for the monitoring of the efficacy of highly active antiretroviral combination therapy. The present report describes reproducible and efficient protocols with enhanced sensitivity for quantitation of HIV-1 RNA from plasma, peripheral blood mononuclear cells, and tissues with Qiagen silica columns for RNA purification combined with the Roche Amplicor HIV-1 Monitor test for quantitative reverse transcription-PCR (RT-PCR). Extraction of RNA from 0.5 ml of plasma resulted in the detection of fewer than 20 HIV RNA copies/ml of plasma, equivalent to the centrifugation-based boosted RT-PCR assay. Silica extraction of cellular RNA resulted in the detection of fewer than 3 HIV-1 RNA copies/microg of total RNA. These techniques facilitate direct comparisons of viral loads between liquid and cellular specimens. Application of these sensitive methods may improve the assessment of the response to new antiretroviral regimens.

DNA Transposable Elements↗
PubMed1991

Expression of defective virus and cytokine genes in murine AIDS.

A syndrome characterized by severe immunodeficiency and lymphoproliferation develops in susceptible strains of mice infected with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV. The etiologic agent in this mixture has been shown to be a replication-defective virus (BM5d) with a 4.8-kb genome that required replication-competent helper viruses, primarily ecotropic (BM5e), for cell-to-cell spread in the host. In the present study, we studied the expression of BM5d and BM5e in tissues of infected mice at various times after inoculation in relation to the expression of cytokine genes that may contribute to the pathogenesis of this disorder. Northern (RNA) analysis of total RNA showed that BM5d was expressed at significant levels in lymphoid tissues within 1 week of infection and that the levels of expression increased with time after inoculation. By 16 weeks postinfection, BM5d was expressed in all tissues examined. Expression of BM5e was relatively more restricted to lymphoid tissues and was detected at lower levels than expression of BM5d at early times after infection, but this virus was expressed in all tissues by 16 weeks. Infection with the virus mixture was associated with constitutive expression of tumor necrosis factor in all tissues examined and of interleukin-1 (IL-1) in lymphoid tissues within 1 week of infection, and at later times with widespread expression of these cytokines and gamma interferon. Also, the levels of interferon regulatory factor 1 mRNA were significantly increased in all infected tissues during the infection. In contrast, expression of IL-3, IL-4, IL-5, and IL-6 was not detectable by Northern analysis of the respective mRNAs in any infected tissue at early or late times postinfection.

Animals↗
PubMed1981

Monocistronic transcription is the physiological mechanism of sea urchin embryonic histone gene expression.

We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.

Animals↗
PubMed1989

Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes.

Production of the mating pheromone alpha-factor was examined in Saccharomyces cerevisiae MAT alpha cells that had been exposed to the mating pheromone a-factor. A 2-h treatment with a-factor caused a significant increase in alpha-factor concentration in the medium as demonstrated by a halo assay. MF alpha 1 is one of the two genes coding for a precursor of alpha-factor. A Northern (RNA) analysis of total RNA from a-factor-treated MAT alpha cells revealed a rapid two- to threefold increase in MF alpha 1 transcript levels, reaching maximum within 60 min of exposure to the pheromone. Pheromone induction did not require ongoing protein synthesis. a-Factor-induced MF alpha 1 expression was quantitated by analysis of an MF alpha 1::SUC2 fusion gene whose product was assayed for invertase activity. Expression of the MF alpha 1::SUC2 gene in MAT alpha cells responded to the a-factor signal like the chromosomal version of MF alpha 1. Maturation of the alpha-factor precursor involves three proteolytic activities which are encoded by the KEX1, KEX2, and STE13 genes, respectively. Two of these genes, namely, KEX2 and STE13, were examined for pheromone-induced expression. Only the STE13 gene exhibited pheromone induction at the transcriptional level.

Blotting, Northern↗
PubMed1993

Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA.

The observation that nuclei from hepatic tissue exhibit specific angiotensin II (Ang II) binding led us to explore whether Ang II modulates mRNA in general, mRNA specific for renin system components, or both. Nuclei from hepatic tissue exhibited a single high-affinity (Kd = 0.4 nmol/L) Ang II-specific binding site, which was associated with increased RNA transcription. Whereas total RNA extracted from nuclei increased 1.5-fold in response to Ang II (10(-9) mol/L), specific mRNA for renin and angiotensinogen increased 7.8- and 2.5-fold, respectively. Ang II binding and induced transcription showed parallel Ang II dose responses that were both inhibited by 10(-5) mol/L DuP 753 or saralasin. Maximum Ang II binding and RNA transcription occurred at the same Ang II concentration (10(-9) mol/L). Higher doses of Ang II resulted in a progressive decrease in RNA transcription. Together, these results demonstrate that hepatic nuclei have functional Ang II-specific receptors. It is concluded that Ang II may elicit responses at nuclear receptors, which heretofore were associated only with Ang II receptors located on plasma membranes. However, the individual contribution of plasma and nuclear membrane Ang II receptors to the overall cellular Ang II transcriptional response and their possible interactions remain to be determined.

Angiotensin II↗
PubMed1997

RNA metabolism in myotonic dystrophy: patient muscle shows decreased insulin receptor RNA and protein consistent with abnormal insulin resistance.

Myotonic dystrophy is a dominantly inherited clinically variable multisystemic disorder, and has been found to be caused by heterozygosity for a trinucleotide repeat expansion mutation in the 3' untranslated region of a protein kinase gene (DM kinase). The mechanisms by which the expanded repeat in DNA results in a dominant biochemical defect and the varied clinical phenotype, is not known. We have recently proposed a model where disease pathogenesis may occur at the RNA level in myotonic dystrophy: the mutant DM kinase RNA with the expansion mutation may disrupt cellular RNA metabolism in some general manner, as evidenced by defects in RNA processing of the normal DM kinase gene in heterozygous patients (dominant negative RNA mutation). Here we further test this hypothesis by measuring RNA metabolism of other genes in patient muscle biopsies (nine adult onset myotonic dystrophy patients, two congenital muscular dystrophy patients, four normal controls, and four myopathic controls). We focused on the insulin receptor gene because of the documented insulin resistance of DM patients. We show that there is a significant decrease in insulin receptor RNA in both total RNA and RNA polyA+ pools relative to normal and myopathic control muscles (P < 0.002), measured relative to both dystrophin RNA and muscle sodium channel RNA. We also show reductions in insulin receptor protein. Our results reinforce the concept of a generalized RNA metabolism defect in myotonic dystrophy, and offer a possible molecular mechanism for the increased insulin resistance observed in many myotonic dystrophy patients.

Adolescent↗
PubMed2006

RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis.

BACKGROUND: Mean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different conditions of exponential growth. RESULTS: It was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes). Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms. CONCLUSION: Our results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and increased transcription of cellular maintenance genes at the expense of growth related genes. The strong link between P:N stoichiometry, RNA:protein ratio, ribosomal requirement for protein synthesis, and growth rate of microorganisms indicated by the study could be used to characterize the N and P economy of complex ecosystems such as soils and the oceans.

Escherichia coli↗
PubMed2004

Regenerative medicine: Antagonic-Stress therapy in distress and aging. I. Preclinical synthesis--2003.

Repetitive and cumulative distress (acute and/or chronic, psychic and/or biologic) and aging processes (impairment phenomena, agglomerated, and accumulated with the passing of life and senescence periods), as well as distress <==> aging reciprocal amplification-accelerating-aggravation relationships require strong and rational (etiopathogenic) therapeutic interventions. Therefore, the drug, Antagonic-Stress (AS)--a new integrative therapy, with specific synergistic formula, being patented worldwide--becomes an important solution in distress, senescence, and their related pathologies. In acute (contention) stress, AS treatment significantly decreased rat mortality, number and surface of stomach ulcerations, nonesterified fatty acids (NEFAs), and increased superoxide dismutase (SOD) from blood. In chronic psychic stress, live nerve cells selectively isolated from rat cerebral cortex were highly protected by the administration of AS. In addition, antistress and antiaging homeostatic actions of AS were demonstrated in accelerated senescence (aging + distress) at multiple brain levels: on functional anabolism [increase in total ribonucleic acids (RNA), total proteins (TP), and water-soluble proteins (WSP)]; on functional catabolism [decrease in water-insoluble proteins (WIP)]; on structural anabolism (increased regeneration of free ribosomes, rough endoplasmic reticulum, mitochondria, and Nissl bodies); on structural catabolism (lipofuscinolysis and ceroidolysis, neurono-glial transfer of lipopigment continuously processed and dissoluted, and finally capillary elimination). Preclinical research with AS demonstrated important regenerative processes in the key organs (liver, heart, and brain), which mostly suffer due to both distress and senescence.

Aging↗
PubMed1977

Preferential inhibition of rDNA transcription by 5-bromodeoxyuridine.

On a chemically defined growth medium the degree of substitution of thymidine with 5-bromodeoxyuridine (BUdR) in DNA of Tetrahymena pyriformis was controlled by the concentration of tetrahydrofiolic acid, BUdR and thymidine in the medium. A correlation between the degree of BUdR substitution in DNA and the reduction in rate of total RNA synthesis has been established. It was found that the reduction of total RNA synthesis results from inhibition of transcription of all RNA species which have been measured. However, independent of the degree of BUdR substitution in DNA, a preferential inhibition of the synthesis of 25s and 17s ribosomal RNA was found. It is concluded that the various genes may respond differently to BUdR substitution with respect to transcription.

Bromodeoxyuridine↗
PubMed2002

[Regulation of Smad7 gene by TGF-beta 1 in process of malignant transformation].

BACKGROUND & OBJECTIVE: Escape from transforming growth factor-beta(TGF-beta)-induced inhibition of growth and proliferation may contribute to tumorigenesis. Smad7 is inhibitory Smads of TGF-beta s signal transduction pathway and prevents TGF-beta signaling. The disorder of Smad7 may lead to the perturbation of TGF-beta signal pathway. In this study, The authors analyzed the expression of Smad7 mRNA and the regulation of Smad7 gene by TGF-beta 1 in the process of malignant transformation of BEP2D cells to investigate the mechanism of cells malignant transformation. METHODS: Cells were cultured and stimulated with TGF-beta 1 followed by RNA extraction. Purified total RNA from TGF-beta 1 treated cells and untreated controls and performed an expression analysis with a human Smad7-specific probe applying Northern blot. As a loading control for the Northern experiment, the membrane was hybridized with a human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) probe. Proteins were extracted from BEP2D and BERP35T-2 cells, then perform Western blot to examine the expression level of TGF-beta 1. RESULTS: Before stimulation with TGF-beta 1, the expression level of Smad7 in the BERP35T-2 cells were higher than that in the BEP2D cells. When stimulated with TGF-beta 1, Smad7 expression levels was upregulated evidently in BEP2D cells, but not significant in BERP35T-2 cells. The expression level of endogenetic TGF-beta 1, BERP35T-2 cells was a little higher than BEP2D cells. CONCLUSION: Over expression of Smad7 mRNA and down-regulation of the cells' responsiveness to TGF-beta 1 in human lung cancer cell line which induced by alpha-particles should be one of the mechanism of radiation induced lung cancer.

Blotting, Western↗
PubMed2003

Expression of messenger ribonucleic acid encoding steroidogenic enzymes in postmenopausal ovaries.

To investigate expression of steroidogenic enzymes involved in androgen and estrogen synthesis in the stroma of postmenopausal ovaries.Ovarian stromal tissue samples were obtained at hysterectomy from postmenopausal women who had hysterectomy and oophorectomy. Tissues were frozen immediately in liquid nitrogen and kept frozen until RNA was extracted. Total RNA was examined by Northern blot analysis using (32)P-labeled cDNA probes encoding human P450 side chain cleavage (P450(SSC)), P450(17alpha), and cytochrome P450 aromatase (P450(arom)). Concentrations of mRNA encoding cytochrome P450(SSC), cytochrome P450 17alpha-hydroxylase (CYP17), and P450(arom) were determined in the ovarian stroma. We detected P450(SSC) mRNA in all postmenopausal ovaries studied. P450(SSC) mRNA was increased threefold in the ovarian stroma of postmenopausal women with endometrial cancer and endometrial hyperplasia. CYP17 mRNA was detected in the ovarian stroma of all women with endometrial cancer. P450(arom) mRNA was not detected in the ovaries studied. Postmenopausal ovaries possess enzymes for initiation of steroidogenesis. Enzymes essential for androgen synthesis were expressed in the ovarian stroma of postmenopausal women with endometrial cancer or hyperplasia.

Aged↗
PubMed1992

Ca pump messenger RNA expression in pregnant rat uterus.

Rat uterus expresses transcripts for the plasma membrane Ca pump PMCA1 and for the sarcoplasmic reticulum Ca pump SERCA2. Uterus contains two mRNA isoforms for PMCA1, one encoding a cyclic nucleotide insensitive Ca pump (PMCA1a) and the other encoding a cyclic nucleotide sensitive pump (PMCA1b). The relative ratio of mRNA to 28S RNA for PMCA1a or PMCA1b and the amount of poly A+ RNA per microgram total RNA obtained from uteri of rats pregnant for 15 days and for parturient rats was similar but the ratio of SERCA2 mRNA to 28S RNA was significantly higher in the parturient rats.

Animals↗
PubMed2003

[Sequence analysis of bacterial transposon in NHX gene of Populus euphratica].

The United Nations Environment Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed. The gene NHX (Na+/H+ exchanger) encodes functional protein that catalyzes the countertransport of Na+ and H+ across membranes and may play an important role in plant salt tolerance. To clone the NHX from the wild plant Populus euphratica collected in Tarim basin and Xinjiang Wujiaqu district into a T-vector, designed primer was used to amplify 1kb NHX cDNA fragment with RT-PCR. Total RNA was extracted from Populus euphratica tissue (plant tissue was collected from Tarim basin and Xinjiang Wujiaqu district and stored in liquid nitrogen) according to the Plant RNA Mini Kits of Omega. First cDNAs were synthesized from 1 microg total RNA of Populus euphratica seedling. A pair of primers were used to perform RT-PCR. The amplified DNA fragment was purified and cloned into pMD18-T vector. However, 1kb and 2.3kb fragment were obtained from Tarim basin and Xinjiang Wujiaqu district and named as PtNHX and PwNHX, respectively. Sequence analysis reveals that the cloned PtNHX fragment of Populus euphratica contains partial NHX coding region with 98%, 86%, 84% and 80% identity comparing with Atriplex gemelini, Suaeda maritima, Arabidopsis thaliana and Oryza sativa, respectively. This analysis suggests that NHX gene would be highly conserved in terms of evolution in plant; and it also suggests that the NHX gene of Populus euphratica also would have the similarity with that of Arabidopsis. It may be of great importance in improvement of the plant salt tolerance and breed of crop. At the same time, sequence analysis shows that PwNHX gene includes a coding region about 1350bp with 99% identity comparing with transposon Tn10 IS10-left transposase of Shigella flexneri. On the one hand, the NHX gene may lose its function because it was inserted a fragment in coding region. On the other hand, its product may play a important role in salt tolerance. Populus grow in saline soil. It speculates that it may have other salt tolerance mechanism in Populus. The transposon can be used as transposon tagging to clone other genes and it will help us to understand farther the salt tolerance mechanism.

Amino Acid Sequence↗
PubMed2006

Genome-wide expression profile of human trabecular meshwork cultured cells, nonglaucomatous and primary open angle glaucoma tissue.

PURPOSE: To contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells to that of control and primary open angle glaucoma (POAG) HTM tissues. METHODS: Cultured HTM cells, HTM tissue dissected from control donors, and HTM tissue from POAG donors receiving medication for glaucoma were fixed in RNA latertrade mark. Total RNA extracted from these samples was linearly amplified with the Ovation Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by quantitative real-time PCR in nonamplified RNA. RESULTS: Cultured HTM cells retained the expression of some genes characteristic of HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants paraoxonase 3 and ceruloplasmin. CONCLUSIONS: Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma.

Case-Control Studies↗
PubMed1999

Two distinct mechanisms mediate a differential regulation of protein kinase C isozymes in acute and prolonged myocardial ischemia.

An activation of protein kinase C (PKC) in acute myocardial ischemia has been shown previously using its translocation to the plasma membrane as an indirect parameter. However, whether PKC remains activated or whether other mechanisms such as altered gene expression may mediate an isozyme-specific regulation in prolonged ischemia have not been investigated. In isolated perfused rat hearts, PKC activity and the expression of PKC cardiac isozymes were determined on the protein level using enzyme activities and Western blot analyses and on the mRNA level using reverse transcriptase-polymerase chain reaction after various periods of global ischemia (1 to 60 minutes). As early as 1 minute after the onset of ischemia, PKC activity is translocated from the cytosol to the particulate fraction without change in total cardiac enzyme activity. This translocation involves all major cardiac isozymes of PKC (ie, PKCalpha, PKCdelta, PKCepsilon, and PKCzeta). This rapid, nonselective activation of PKCs is only transient. In contrast, prolonged ischemia (>/=15 minutes) leads to an increased cardiac PKC activity (119+/-7 versus 190+/-8 pmol/min per mg protein) residing in the cytosol. This is associated with an augmented, subtype-selective isozyme expression of PKCdelta and PKCvarepsilon (163% and 199%, respectively). The specific mRNAs for PKCdelta (948+/-83 versus 1501+/-138 ag/ng total RNA, 30 minutes of ischemia) and PKCepsilon (1597+/-166 versus 2611+/-252 ag/ng total RNA) are selectively increased. PKCalpha and PKCzeta remain unaltered. In conclusion, two distinct activation and regulation processes of PKC are characterized in acute myocardial ischemia. The early, but transient, translocation involves all constitutively expressed cardiac isozymes of PKC, whereas in prolonged ischemia an increased total PKC activity is associated with an isozyme-selective induction of PKCepsilon and PKCdelta. Whether these fundamentally different activation processes interact remains to be elucidated.

Acute Disease↗