Up-regulation of nuclear PLCbeta1 in myogenic differentiation

Phospholipase C beta(1) (PLCbeta(1)) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCbeta(1), i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCbeta(1)a and PLCbeta(1)b. Indeed, treatment with insulin induces a dramatic rise of both PLCbeta(1) subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti-PLCbeta(1) specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCbeta(1) expression versus Troponin T expression clearly indicates that the increase in the amount of PLCbeta(1) takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCbeta(1)M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCbeta(1) is a key player in myoblast differentiation, functioning as a positive regulator of this process.     

Effects of intrahippocampal CT105, a carboxyl terminal fragment of beta-amyloid precursor protein,alone/with inflammatory cytokines on working memory in rats

In this study, we examined the effects of a 105 amino acid carboxyl terminal fragment of beta-amyloid precursor protein (CT105) and inflammatory cytokines on working memory in rats, by using a three-panel runway set-up. CT105 at 10 nmol/side significantly impaired working memory when it was administered bilaterally into the hippocampus. Furthermore, to elucidate the interaction of CT105 with inflammatory cytokines, we co-administered tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in combination with CT105. Concurrent injections of CT105 (1.0 nmol/side) and TNF-alpha (100 ng/side) produced a synergistic deficit of working memory, whereas IL-1beta (100 ng/side) combined with CT105 (1.0 nmol/side) did not affect the working memory performance. These results indicate that the CT105-induced impairment of working memory is strongly aggravated by an increase in the level of the inflammatory cytokine TNF-alpha, which may occur in the brains of patients with Alzheimer's disease.     

Metabolic and cardiorespiratory responses to "the lactate clamp"

To evaluate the hypothesis that precursor supply limits gluconeogenesis (GNG) during exercise, we examined training-induced changes in glucose kinetics [rates of appearance (R(a)) and disappearance (R(d))], oxidation (R(ox)), and recycling (R(r)) with an exogenous lactate infusion to 3.5-4.0 mM during rest and to pretraining 65% peak O(2) consumption (VO(2 peak)) levels during exercise. Control and clamped trials (LC) were performed at rest pre- (P(R)R, P(R)R-LC) and posttraining (P(O)R, P(O)R-LC) and during exercise pre- (P(R)E(X)) and posttraining at absolute (P(O)A(B), P(O)A(B)-LC) and relative (P(O)R(L), P(O)R(L)-LC) intensities. Glucose R(r) was not different in any rest or exercise condition. Glucose R(a) did not differ as a result of LC. Glucose R(ox) was significantly decreased with LC at P(O)R (0.38 +/- 0.03 vs. 0.56 +/- 0.04 mg. kg(-1). min(-1)) and P(O)A(B) (3.82 +/- 0.51 vs. 5.0 +/- 0.62 mg. kg(-1). min(-1)). Percent glucose R(d) oxidized decreased with all LC except P(O)R(L)-LC (P(R)R, 32%; P(R)R-LC, 22%; P(O)R, 27%; P(O)R-LC, 20%; P(O)A(B), 95%; P(O)A(B)-LC, 77%), which resulted in a significant increase in oxidation from alternative carbohydrate (CHO) sources at rest and P(O)A(B). We conclude that 1) increased arterial [lactate] did not increase glucose R(r) measured during rest or exercise after training, 2) glucose disposal or production did not change with increased precursor supply, and 3) infusion of exogenous CHO in the form of lactate resulted in the decrease of glucose R(ox).     

mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.     

Major gene evidence after MTHFR-segregation analysis of serum homocysteine in families of patients undergoing coronary arteriography

Elevated levels of homocysteine is a risk factor for coronary artery disease. Polymorphic alleles in the MTHFR genes that cause recessively inherited increased homocysteine level can explain only a small proportion of the observed variation in homocysteine level. To investigate additional genetic influences, we examined environmental, familial, and genetic influences on serum homocysteine levels in 661 family members of 112 probands who underwent elective coronary arteriography. Maximum likelihood methods were used to fit several genetic and non-genetic models of inheritance to these data to determine if an unobserved Mendelian major gene could explain the familial homocysteine distribution. Adjustments for age, lifestyle (smoking and alcohol consumption), serum folate and vitamin B12, and the measured genotype effect of the MTHFR C677T mutation was carried out separately for males and females using multiple regression models for homocysteine, before and after log-transformation prior to this segregation analysis. After excluding the effects of mutations in the MTHFR genes, we found evidence of a major gene acting in a co-dominant manner. Estimated mean homocysteine levels for the three putative genotypes (LL, LH, and HH) were 8.0, 10.1, and 15.9 micro mol/l, respectively, with relative frequencies of 56.8%, 37.2%, and 6%, respectively. Our analysis suggested the presence of a co-dominantly expressed major gene, in addition to the effects of the MTHFR C677T mutation. The results of this study also indicated that multifactorial inheritance was supported more strongly than Mendelian inheritance alone. Our findings may have implications for attempts to identify new homocysteine susceptible genes.     

Optimization of submerged culture conditions for the production of angiotensin converting enzyme inhibitor from <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The angiotensin-converting enzyme (ACE) inhibitory effect was tested in the culture broth from submerged mycelial cultures of 20 basidiomycetes. The ACE inhibitory effect of culture broth from Flammulina velutipes strain 414 was the highest (52.8%), followed by Lentinus edodes strains 2 (44.4%) and 16 (41.3%). Nutritional requirements for the production of ACE inhibitory substance from F. velutipes were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of ACE inhibitory substance. The optimal medium composition was (g/l): sucrose 20, ammonium acetate 5, glutamic acid 2, KH₂PO₄ 3, MgSO₄·7H₂O 0.8, and yeast extract 0.5. Under optimal culture conditions, the ACE inhibitory effect was more than 80%. Received 04 May 2002/ Accepted in revised form 11 June 2002     

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.     

A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.