Biomedical subjects

L Wei

Publications and source records attributed to L Wei.

At least 19 recordsLinked to original sources

PubMed2001

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Animals↗

PubMed2001

Partial imputation approach to analysis of repeated measurements with dependent drop-outs.

In clinical trials repeated measurements of a response variable are usually taken at prespecified time-points to compare the treatment effects. However, the comparison of treatment effects is often complicated by missing data caused by the withdrawal of some patients before the end of the study (that is, drop-outs). When the drop-out process depends on the response variable of interest, ignoring missing data may lead to biased comparison of the treatment effect. In this paper, conditions for ignoring the dependent missingness are investigated and a new approach using the usual testing procedure based on data with partial carrying-forward imputation is proposed. The proposed approach is conceptually and practically simple, and is motivated by making incremental improvement on the familiar 'all available data' (AAD) approach and the 'last value carrying forward' (LVCF) approach, which are commonly used in data analysis with drop-outs by practitioners. It is also compared favourably to the mixed-effect model approach with dependent drop-outs. Simulations and real data are used to evaluate and illustrate statistical properties of the proposed approach. The principle of the proposed approach can also be extended to using other imputation methods such as the multiple imputation.

Analysis of Variance↗

PubMed2001

Tyrosine kinase-dependent, phosphatidylinositol 3'-kinase, and mitogen-activated protein kinase-independent signaling pathways prevent lung adenocarcinoma cells from anoikis.

Normal epithelial cells are anchorage-dependent. Detachment of normal epithelial cells from their substratum causes apoptosis, termed anoikis. Malignant tumor cells, however, can survive and proliferate independent of anchorage. To understand the molecular basis of tumor cell anchorage independence, we investigated the role of tyrosine kinases and their downstream signaling pathways in anoikis resistance of human lung adenocarcinoma cells. Four of the five lung adenocarcinoma cell lines analyzed are resistant to anoikis. Tyrosine kinase inhibitor genistein rendered three of them sensitive to anoikis. Although cell detachment induced rapid protein tyrosine dephosphorylation in Madin-Darby canine kidney cells, a nontransformed epithelial cell line, tyrosine phosphorylation of several proteins in the tumor cells is anchorage-independent. Similarly, phosphorylation of Akt and mitogen-activated protein kinase, two signaling proteins downstream of tyrosine kinases, was decreased in Madin-Darby canine kidney cells but increased in some of the tumor cells upon cell detachment. Inhibition of phosphorylation of the two proteins, however, did not induce anoikis in the tumor cells. Specific inhibitors to several known tyrosine kinases also failed to induce anoikis in these cells. These data suggest the existence of tyrosine kinase-dependent phosphatidylinositol 3'-kinase and mitogen-activated protein kinase-independent signaling pathways that function to regulate cell survival and death. Alteration in these pathways may count for the anchorage-independent survival of the lung adenocarcinoma cells and other malignant tumor cells.

Adenocarcinoma↗

PubMed2001

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex.

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA-DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA-DNA substrate. Binding of NS3 to the PNA-DNA substrate was similar to the DNA-DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA-RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino-DNA and phosphorothioate-DNA substrates were utilized as efficiently by NS3 as DNA-DNA substrates. These results indicate that the PNA-DNA and PNA-RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.

Catalysis↗

PubMed2001

A modified ex vivo skin organ culture system for functional studies.

To investigate the immunological function of cells in normal and diseased skin under conditions approximating the in vivo situation, it is necessary to maintain the structural integrity of the tissue. To achieve this, freshly isolated skin has to be cultured ex vivo, or an in vitro-constructed complete skin equivalent may be used. Different skin organ culture systems have been described. Basically two systems prevail: submerged or air-exposed skin organ cultures. The former model has been used for measuring cytokine secretion by skin cells in the medium, and the latter to study the expression of cell membrane proteins in situ and the kinetics of epidermal Langerhans cells. Here we present a modified ex vivo skin organ culture system which approaches the in vivo situation by maintaining the normal skin architecture without spontaneous induction of the regenerative maturation markers. This method allowed the expression of cell membrane proteins in situ to be measured, and the cytokine secretion by skin cells in the culture medium to be quantitated in the same experiment. In this system, both general and specific stimuli (LPS and IL-1beta) upregulated the expression of skin-derived cytokines IL-8 and IL-6 in the medium and different markers (ICAM-1, CD40 and CD86) on cells in the tissue in a 24-hour culture-formed. Elevation of both cytokine and cell marker expression could be blocked by dexamethasone and by IL-1ra which acts specifically on IL-1beta-mediated responses. The system presented here is both quick and simple and can be used as a model to study the behaviour of skin cells in their natural microenvironment.

Antigens, CD↗

PubMed2001

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Amino Acid Motifs↗

PubMed2001

Lead-induced hypertension. III. Increased hydroxyl radical production.

Lead-induced hypertension has previously been shown to be closely associated with an increase in reactive oxygen species in low lead (100 ppm)-treated rats. The present study has attempted to define the specific moiety involved by noting the blood pressure (BP), reactive oxygen species (MDA-TBA), hydroxyl radical, and nitrotyrosine responses to infusion of the reactive oxygen species scavenger dimethylthiourea. Dimethylthiourea, a reputed scavenger of hydroxyl radical, normalized BP and MDA-TBA in the lead-treated rats but had no effect in normal control animals. MDA-TBA, hydroxyl radical, and nitrotyrosine, the tissue end product of peroxynitrite, were reduced to or toward normal by dimethylthiourea. The results, therefore, are consistent with the suggestion that either hydroxyl radical or peroxynitrite may be the reactive species affected by lead.

Animals↗

PubMed2001

The cytotoxic effect of E1B 55-kDa mutant adenovirus on human hepatocellular carcinoma cell lines.

It has been suggested the E1B 55 kDa mutant adenovirus dl1520 can selectively kill p53-deficient human tumor cells. In this study, we examined the cytotoxic effect of dl1520 on nine human hepatocellular carcinoma (HCC) cell lines with different p53 genetic and functional status. The results showed that HCC cell lines with deleted or mutant p53 gene and reduced p53 transcriptional activities were more susceptible to dl1520-induced cytolysis. Hep3B (p53-null) and HepG2 (p53-wt) cells were arrested at G2/M phase when cytolysis occurred. Cyclin-dependent kinase inhibitor (CDKI) p21(Waf-1/Cip-1) was downregulated 24 hours after dl1520 infection in HepG2 cells and increased when cytolysis occurred. No p21 expression was detected in Hep3B cells. DNA fragmentation was found in both Hep3B and HepG2 cells after dl1520 infection. Bax expression increased in dl1520-infected HepG2 cells but not in Hep3B cells. Notably, three Bax-like proteins, molecular mass around 40 to 80 kDa, accumulated 48 hours after adenovirus infection in Hep3B cells but not in HepG2 cells. These results suggest that the susceptibility of HCC cells to dl1520-induced cytolysis is related to both p53 genotype and functional status, and is mediated by both cell cycle disturbance and apoptosis.

Adenoviridae↗

PubMed2001

Association of parvovirus B19 infection with idiopathic collapsing glomerulopathy.

BACKGROUND: Collapsing glomerulopathy (CG), a disorder with severe glomerular and tubular involvement, occurs either as an idiopathic lesion or in some patients with human immunodeficiency virus (HIV) infection known as HIV-associated nephropathy (HIVAN). We previously reported a renal transplant recipient with de novo CG and red cell aplasia in association with persistent parvovirus B19 (PVB19) infection. This prompted us to look for an association between PVB19 infection and CG. METHODS: DNA from archived biopsies of patients with CG was analyzed for PVB19 by polymerase chain reaction (PCR). Results were compared with HIVAN, idiopathic focal segmental glomerulosclerosis (FSGS), and controls. In situ hybridization (ISH) was done to localize PVB19 in renal biopsies. Peripheral blood specimens of patients with CG, HIV infection, healthy controls, and randomly selected hospitalized patients (sick controls) were also analyzed for PVB19. RESULTS: PVB19 DNA was detected in renal biopsies of 18 out of 23 (78.3%) patients with CG, 3 out of 19 (15.8%) with HIVAN, 6 out of 27 (22.2%) with FSGS, and 7 out of 27 (25.9%) controls (P < 0.01, CG vs. HIVAN, FSGS, and controls). PVB19 was detected in peripheral blood of 7 out of 8 (87.5%) CG patients, 3 out of 22 (13.6%) with HIV infection, 4 out of 133 (3%) healthy controls, and 2 out of 50 (4%) sick controls (P < 0.001, CG vs. HIV infected, healthy, and sick controls). PVB19 was identified in glomerular parietal and visceral epithelial and tubular cells by ISH. CONCLUSIONS: The significantly higher prevalence of PVB19 DNA in renal biopsies and peripheral blood of CG patients suggests a specific association between PVB19 infection and CG. In susceptible individuals, renal epithelial cell infection with PVB19 may induce CG.

Biopsy↗

PubMed2001

Effect of load on articular cartilage matrix and the development of guinea-pig osteoarthritis.

OBJECTIVE: To study the biochemical changes in the early development of primary guinea-pig knee osteoarthritis (OA) and its dependence on load. METHODS: Load distribution was modified with below-knee amputation or femur valgus osteotomy in 9-month-old guinea-pigs. Soft tissue sham operated animals served as controls. The composition of uncalcified and calcified articular cartilage at the medial and lateral tibial condyle was studied by analysing small and large proteoglycans (PG) by gel electrophoresis and sulfation pattern with high-performance liquid chromatography. Collagen concentration was also determined. RESULTS: The articular surfaces with a presumed higher load after surgery had a slight, but consistent, higher water content. Decreased load-on the ipsilateral medial condyle after femur osteotomy, and on the ipsilateral medial and lateral condyles following tibia amputation-was associated with an increased concentration of PGs, while this concentration decreased in condyles with increased load. Collagen concentration followed a similar pattern in the osteotomy group. In the amputated animals collagen concentration went down in all condyles, regardless of change of load. The aggregability and proportion of large and small PGs, the concentration of hyaluronan and the sulfation pattern of chondroitin sulfate was not affected by load. No consistent changes in PG, collagen or HA concentration, HA aggregability or sulfation pattern were seen in the calcified cartilage. CONCLUSIONS: Primary guinea-pig knee OA is a reproducible model similar to human OA. It develops slowly and biochemical changes seem to appear before the morphological lesions become evident. The biochemical events are affected by load redistribution and correlate closely to morphological changes. These changes eventually result in a cartilage devoid in aggrecan, as also has been demonstrated in advanced human OA. All of this makes primary guinea-pig OA a suitable model for studying early OA changes.

Animals↗

PubMed2001

beta1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal alpha-actin promoter.

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G-actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differentiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF-dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA-dependent skeletal alpha-actin promoter activation requires beta1 integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activation of the alpha-actin promoter by RhoA is greatly potentiated (up to 15-fold) by co-expression of the integrin beta1A or beta1D isoform but is significantly reduced by 70% with a co-expressed dominant negative mutant of beta1 integrin. Furthermore, clustering of beta1 integrin with anti-beta1 integrin antibodies potentiates synergistic RhoA and beta1 integrin activation of the alpha-actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA-induced activation of the alpha-actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and beta1 integrin on the actin promoter. These observations support the concept that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with beta1 integrin signal pathway via an organized actin cytoskeleton.

3T3 Cells↗

PubMed2001

Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase.

The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

Amino Acid Sequence↗

PubMed2001

Using meta computing tools to facilitate large-scale analyses of biological databases.

Given the high rate at which biological data are being collected and made public, it is essential that computational tools be developed that are capable of efficiently accessing and analyzing these data. High-performance distributed computing resources can play a key role in enabling large-scale analyses of biological databases. We use a distributed computing environment, Legion, to enable large-scale computations on the Protein Data Bank (PDB). In particular, we employ the Feature program to scan all protein structures in the PDB in search for unrecognized potential cation binding sites. We evaluate the efficiency of Legion's parallel execution capabilities and analyze the initial biological implications that result from having a site annotation scan of the entire PDB. We discuss four interesting proteins with unannotated, high-scoring candidate cation binding sites.

Algorithms↗

PubMed2001

Dexamethasone inhibits the stimulation of muscle protein synthesis and PHAS-I and p70 S6-kinase phosphorylation.

Glucocorticoids inhibit protein synthesis in muscle. In contrast, insulin and amino acids exert anabolic actions that arise in part from their ability to phosphorylate ribosomal p70 S6-kinase (p70(S6k)) and eukaryotic initiation factor (eIF)4E binding protein (BP)1 (PHAS-I), proteins that regulate translation initiation. Whether glucocorticoids interfere with this action was examined by giving rats either dexamethasone (DEX, 300 microg. kg(-1). day(-1), n = 10) or saline (n = 10) for 5 days. We then measured the phosphorylation of PHAS-I and p70(S6k) in rectus muscle biopsies taken before and at the end of a 180-min infusion of either insulin (10 mU. min(-1). kg(-1) euglycemic insulin clamp, n = 5 for both DEX- and saline-treated groups) or a balanced amino acid mixture (n = 5 for each group also). Protein synthesis was also measured during the infusion period. The results were that DEX-treated rats had higher fasting insulin, slower glucose disposal, less lean body mass, and decreased protein synthetic rates during insulin or amino acid infusion (P < 0.05 each). DEX did not affect basal PHAS-I or p70(S6k) phosphorylation but blocked insulin-stimulated phosphorylation of PHAS-I- and amino acid-stimulated phosphorylation of both PHAS-I and p70(S6k) (P < 0.01, for each). DEX also increased muscle PHAS-I concentration. These effects can, in part, explain glucocorticoid-induced muscle wasting.

Amino Acids↗

PubMed2001

Branched chain amino acids activate messenger ribonucleic acid translation regulatory proteins in human skeletal muscle, and glucocorticoids blunt this action.

Branched chain amino acids (BCAA) are particularly effective anabolic agents. Recent in vitro studies suggest that amino acids, particularly leucine, activate a signaling pathway that enhances messenger ribonucleic acid translation and protein synthesis. The physiological relevance of these findings to normal human physiology is uncertain. We examined the effects of BCAA on the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (eIF4E-BP1) and ribosomal protein S6 kinase (p70(S6K)) in skeletal muscle of seven healthy volunteers. We simultaneously examined whether BCAA affect urinary nitrogen excretion and forearm skeletal muscle protein turnover and whether the catabolic action of glucocorticoids could be mediated in part by inhibition of the action of BCAA on the protein synthetic apparatus. BCAA infusion decreased urinary nitrogen excretion (P < 0.02), whole body phenylalanine flux (P < 0.02), plasma phenylalanine concentration (P < 0.001), and improved forearm phenylalanine balance (P = 0.03). BCAA also increased the phosphorylation of both eIF4E-BP1 (P < 0.02) and p70(S6K) (P < 0.03), consistent with an action to activate the protein synthetic apparatus. Dexamethasone increased plasma phenylalanine concentration (P < 0.001), prevented the BCAA-induced anabolic shift in forearm protein balance, and inhibited their action on the phosphorylation of p70(S6K). We conclude that in human skeletal muscle BCAA act directly as nutrient signals to activate messenger ribonucleic acid translation and potentiate protein synthesis. Glucocorticoids interfere with this action, and that may be part of the mechanism by which they promote net protein catabolism in muscle.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Effects of an angelica extract on human erythrocyte aggregation, deformation and osmotic fragility.

In Chinese traditional medicine, angelica is widely used for its known clinical effects of ameliorating blood microcirculation. But the mechanism of these beneficial effects still remains unclear. In this work the rheological behaviour of human erythrocytes treated by angelica was studied in vitro. Normal RBCs incubated with an angelica extract at different concentrations (5, 10 or 20 mg/ml) for 60 min at 37 degrees C and then their aggregation, deformation and osmotic fragility were measured with different recently developed optical techniques, namely Erythroaggregometer (Regulest, Florange, France), LORCA (Mechatronics, Amsterdam) and Fragilimeter (Regulest, Florange, France). Experimental results show that angelica (20 mg/ml) significantly decreased normal RBCs' aggregation speed (p<0.01) and could inhibit the hyperaggregability caused by dextran 500. However, the strength of normal RBCs aggregates were not influenced by angelica. When a calcium ionophore A23187 (1.9 microM) was used to harden cell membrane, angelica (20 mg/ml) could significantly (p<0.01) protect erythrocytes against the loss of their deformability even it had no effects on normal RBCs deformation. Finally angelica (5 and 10 mg/ml) decreased significantly (p<0.01) normal RBCs osmotic fragility. In conclusion angelica plays a rheologically active role on human erythrocytes, and this study suggests a possible mechanism for angelica's positive effects against certain cardiovascular diseases.

Asteraceae↗

PubMed2000

Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone.

Parathyroid hormone (PTH) and its related peptide regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. However, the intracellular pathway for transducing PTH/PTH-related peptide signals in chondrocytes remains unclear. Here, we show that this pathway is mediated by mitogen-activated protein kinase (MAPK) p38. Incubation of hypertrophic chondrocytes with PTH (1-34) induces an inhibition of p38 kinase activity in a time- and dose-dependent manner. Inhibition of protein kinase C prevents PTH-induced p38 MAPK inhibition, whereas inhibition of protein kinase A has no effect. Thus, protein kinase C, but not protein kinase A, is required for the inhibition of p38 MAPK by PTH. Treatment of hypertrophic chondrocytes by PTH or by p38 MAPK inhibitor SB203580 up-regulates Bcl-2, suggesting that Bcl-2 lies downstream of p38 MAPK in the PTH signaling pathway. Inhibition of p38 MAPK in hypertrophic chondrocytes by either PTH, SB303580, or both together leads to a decrease of hypertrophic marker type X collagen mRNA and an increase of the expression of prehypertrophic marker cartilage matrix protein. Therefore, inhibition of p38 converts a hypertrophic cell phenotype to a prehypertrophic one, thereby preventing precocious chondrocyte hypertrophy. Taken together, these data suggest a major role for p38 MAPK in transmitting PTH signals to regulate chondrocyte differentiation.

Animals↗

PubMed2000

The ubiquitin-proteasome proteolytic pathway in heart vs skeletal muscle: effects of acute diabetes.

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P </= 0.05). This suggests that the ubiquitin proteasomal pathway may also be important for normal heart muscle turnover. Diabetes increased ubiquitin mRNA by approximately 50% in heart (P < 0.03) and by approximately 100% in skeletal muscle (P < 0.005). It remained high after 3 days of insulin treatment in both tissues. C2 and C8 subunit mRNA did not change with diabetes or insulin treatment. Diabetes did not change the amount of free ubiquitin or the proteasomal (lactacystin-inhibitable) chymotrypsin-like peptidase activity in heart or skeletal muscle. In conclusions, gene expression for several components of the ubiquitin-proteasome proteolytic pathway is significantly higher in cardiac than in skeletal muscle, as is the proteasome chymotrypsin-like peptidase activity. Diabetes increases the expression of ubiquitin but not C2 or C8 subunit mRNA, nor does it significantly alter the amount of free ubiquitin or the proteasome chymotrypsin-like peptidase activity. The rate-limiting step of enhanced protein degradation in diabetic rat heart and skeletal muscle may be located at ubiquitin conjugation and/or its binding to proteasome, not at the ubiquitin availability or the proteasome itself.

Acute Disease↗