Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Structural and functional studies on Troponin I and Troponin C interactions

Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.     

Heat shock mediated modulation of protein kinase CK2 in the nuclear matrix

Nuclear matrix, a key structure in the nuclear framework, appears to be a particularly responsive target during heat shock treatment of cells. We have previously shown that nuclear matrix is a preferential target for protein kinase CK2 signaling in the nucleus. The levels of CK2 in the nuclear matrix undergo dynamic changes in response to altered growth status in the cell. Here, we have demonstrated that CK2 targeting to the nuclear matrix is profoundly influenced by treatment of the cells to temperatures higher than 37 degrees C. Rapid increase in the nuclear matrix association of CK2 is observed when cells are placed at temperatures of 41 and 45 degrees C. This effect at 45 degrees C was higher than at 41 degrees C, and was time-dependent. Also, different cell lines behaved in a qualitatively similar manner though the quantitative responses differed. The modulations in the nuclear matrix associated CK2 in response to heat shock appear to be due to trafficking of the enzyme between cytosolic and nuclear compartments. In addition, it was noted that isolated nuclei subjected to heat shock also responded by a shuttling of the intrinsic CK2 to the nuclear matrix compartment. These results suggest that modulations in CK2 in the nuclear compartment in response to the heat stress occur not only by a translocation of the enzyme from the cytoplasmic compartment to the nuclear compartment, but also that there is a redistribution of the kinase within the nuclear compartment resulting in a preferential association with the nuclear matrix. The results support the notion that CK2 association with the nuclear matrix in response to heat shock may serve a protective role in the cell response to stress.     

Computerized automated morphometric assay including frequency estimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis

An in vivo study of the effects of pentachlorophenol was carried out with a pre-acclimatized fish species, Heteropneustes fossilis, using four sub-lethal concentrations, 0.1, 0.2, 0.3 and 0.4 ppm, and three sampling times, 48, 72 and 96 h. Cytogenetic preparations were stained by the haematoxylin-eosin technique. The incidence of micronuclei was scored by a manual and an automated method. Small-sized micronuclei appeared in the cytoplasm in addition to the main nucleus. The frequency of micronucleated erythrocytes peaked at 4 days (96 h) exposure. The percentage of single micronuclei increased with longer exposures. The Mann-Whitney U test showed all micronuclei frequencies were significantly different from control (P<0.05). No statistical difference was observed between scores obtained by the manual and automated methods. A linear relationship between the percentage of micronucleated erythrocytes and dose was confirmed at all levels. Computer image analysis of morphological variations of erythrocytes indicated a 1:5 ratio of micronuclei and main nucleus accompanied by a reduction in cell volume by 600 dot units. Pentachlorophenol-mediated genotoxicity was confirmed in this fish for the first time. Possible consequences of genotoxicity and cytotoxicity are discussed.     

Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Production and characterization of cellulolytic and xylanolytic enzymes from the ligninolytic white-rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse

The ligninolytic white-rot fungus Phanerochaete chrysosporium BKM-F-1767 produced extracellular cellulolytic enzymes (carboxymethylcellulase, CMCase and -glucosidase) and xylanolytic enzymes (xylanase and -xylosidase) in liquid medium containing 1.0% sugarcane bagasse with or without 1.0% glucose. The changes in pH and soluble protein content were monitored in the culture filtrates. The results obtained showed that the pH decreased after 3 days and then increased. The soluble protein content increased and reached the maximum value after 12 days. The results showed that the activities of enzymes were higher in the case of sugarcane bagasse without glucose. The characterization study indicated that the optimum pH values were 4.6, 4.2, 5.0 and 5.0 for CMCase, -glucosidase, xylanase and -xylosidase, respectively and the optimum temperatures were 60, 70, 65 and 60 °C for the investigated enzymes, respectively. The results showed also that after prolonged heating (5 h) at 60 °C, CMCase, -glucosidase, xylanase and -xylosidase retained 81.2, 86.8, 51.5 and 27.4% activity, respectively.