The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Lactate racemase. Hydroxylamine-dependent 18O exchange of the alpha-hydroxyl of lactic acid.

The lactic acid racemase (EC 5.1.2.1) derived from Clostridium butylicum catalyzes the racemization of the alpha-18O label. The proposed alpha-carbonyl intermediate for the enzyme-catalyzed reaction has been previously shown to be trapped as an enzyme-bound oxime in the presence of hydroxylamine. This report demonstrates that the formation of the inactive enzyme-bound oxime, followed by reactivation in the presence of an excess of competing free carbonyl (pyruvic acid) results in a complete loss of the alpha-18O label from an original alpha-18O-labeled lactic acid.     

The biosynthesis of rat serum albumin. Metabolism of the NH2-terminal hexapeptide of proalbumin

Proalbumin differs from serum albumin in containing a leading hexapeptide segment, Arg-Gly-Val-Phe-Arg-Arg. This propeptide is removed in the Golgi complex immediately prior to secretion of the albumin, but its fate and possible functions are unknown. We have tested for the presence of the propeptide and its immediate catabolic products in rat liver and plasma and have studied both the disappearance of 3H-propeptide after intravenous injection and the breakdown of synthetic propeptide by rat liver cell components and plasma in vitro. We found no detectable propeptide or its two pentapeptide derivatives in rat liver or plasma at a sensitivity of less than 1 microM. Injected 3H-propeptide was completely cleared from blood within 2 min. No binding of free propeptide to serum albumin was observed. Liver cell fractions as well as blood plasma degraded added propeptide, with the highest activity being observed in smooth microsomes, the Golgi-enriched fraction, and plasma membrane. These preparations chiefly removed the terminal arginine residues, whereas enzymes in the cytosol degraded the peptide completely to amino acids. The activity in plasma resided largely in an alpha-globulin with molecular mass of about 280,000 Da which appears to be carboxypeptidase N. We conclude that the liberated propeptide is quickly broken down within the liver cell and does not accumulate in an amount sufficient to exert feedback or other effects on albumin synthesis.     

Prevalence and identity of coccidia in pen-raised wild turkeys

One hundred nineteen pen-raised wild turkeys (Meleagris gallopavo) from 12 locations in nine states in the United States were examined for coccidia by sugar flotation of intestinal contents and mucosa or by subinoculating the contents into uninfected domestic turkeys. Seventy-eight (66%) of the turkeys were positive for coccidia. There were no differences in the frequency of coccidia among adult, sub-adult or juvenile turkeys. More females (75%) were infected than males (48%). The species of coccidia from 30 of the turkeys were identified based on microscopic examination of oocysts, fresh scrapings, stained sections and inoculations of bobwhites (Colinus virginianus). The frequency of each species was Eimeria meleagrimitis (97%), E. gallopavonis (47%), E. meleagridis (27%), E. dispersa (17%), E. innocua-E. subrotunda (13%), E. adenoeides (7%) and an undescribed species (3%). Of the 30 turkeys in which the species of coccidia was determined, 30% had a single species infection, 40% had two species, 20% had three species and 10% had four species.     

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.