Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.     

Trichophyton indotineae sp. nov.: A New Highly Terbinafine-Resistant Anthropophilic Dermatophyte Species

Mycopathologia - In this report, we describe the first isolation of two highly terbinafine (TRF)-resistant Trichophyton interdigitale-like strains from a Nepali patient and an Indian patient with...     

Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.     

Effect of Amino Acids on Lysosomal Proteolytic Activities in Rat Livers

(R)-2-(4′-Isobutylphenyl)propanoic acid (ibuprofen), (S)-3(4′-isobutylphenyl)butanoic acid and (S)-4-(4′-isobutylphenyl)pentanoic acid were obtained using microbial oxidation of (±)-l-isobutyl-4-(1′ -methyloctyl)benzene by Rhodococcus sp. BPM 1613.     

Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia

The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, we disrupted these genes in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis.     

Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs.

We previously reported that the cytosolic phospholipase A(2) (cPLA2) pathway is involved in ventilator-induced lung injury (VILI) produced by high peak inflation pressures (PIP) (J Appl Physiol 98: 1264-1271, 2005), but the relative contributions of the various downstream products of cPLA2 on the acute permeability response were not determined. Therefore, we investigated the role of cPLA2 and the downstream products of arachidonic acid metabolism in the high-PIP ventilation-induced increase in vascular permeability. We perfused isolated mouse lungs and measured the capillary filtration coefficient (K(fc)) after 30 min of ventilation with 9, 25, and 35 cmH2O PIP. In high-PIP-ventilated lungs, K(fc) increased significantly, 2.7-fold, after ventilation with 35 cmH2O PIP compared with paired baseline values and low-PIP-ventilated lungs. Also, increased phosphorylation of lung cPLA2 suggested enzyme activation after high-PIP ventilation. However, treatment with 40 mg/kg arachidonyl trifluoromethyl ketone (an inhibitor of cPLA2) or a combination of 30 microM ibuprofen [a cyclooxygenase (COX) inhibitor], 100 microM nordihydroguaiaretic acid [a lipoxygenase (LOX) inhibitor], and 10 microM 17-octadecynoic acid (a cytochrome P-450 epoxygenase inhibitor) prevented the high-PIP-induced increase in K(fc). Combinations of the inhibitors of COX, LOX, or cytochrome P-450 epoxygenase did not prevent significant increases in K(fc), even though bronchoalveolar lavage levels of the COX or LOX products were significantly reduced. These results suggest that multiple mediators from each pathway contribute to the acute ventilator-induced permeability increase in isolated mouse lungs by mutual potentiation.