Cultured peritoneal mesothelial cells exhibit apical primary cilia

This study examined primary cilia on cultured human and rabbit peritoneal mesothelial cells (PMC) and investigated factors that influence ciliary expression. Primary cilia were examined with indirect immunocytochemistry, laser scanning confocal microscopy and scanning electron microscopy. Ciliary expression was evaluated in cultures with or without l-cysteine (0.25 mM) or exposure to Ca(2+)-free Krebs-Ringer solution supplemented with EGTA, 0.5 mM. This treatment disrupted cell monolayer integrity. Cilia were counted and normalized to total cell counts using NIH image. Primary cilia were identified on both human and rabbit PMC. Cells treated with l-cysteine expressed significantly more cilia compared with monolayers deprived of l-cysteine. Exposure to Ca(2+)-free Krebs-Ringer solution significantly reduced cilia (5.9+/-1.0%, n=7). Although ciliary expression could be augmented with l-cysteine, approximately 60% of human PMC and 84% of rabbit PMC did not exhibit cilia. Together, these data show that monolayers of PMC express apical cilia that can be augmented with l-cysteine, independently of increased cell density.     

Non-enzymatic and microsome-dependent binding of polycyclic hydrocarbons to DNA and polynucleotides

The binding of tritium-labelled 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and 3-methylcholanthrene (MCA) to DNA or polynucleotides in vitro was re-examined both in the presence and in the absence of rat liver or human placental microsomes.A high level of non-enzymatic binding was evident when thymus DNA was used as acceptor. This non-enzymatic binding made it difficult to determine the effect of microsomes, except in the case of BP when induced rat microsomes were used. Better results were obtained using polynucleotides: a definite microsome-dependent binding occurred between all the polynucleotides and all the hydrocarbons tested.No clear evidence of binding catalysed by microsomes from human placenta was found except in polynucleotide-BP interactions: further studies are required to completely evaluate the ability of such nucleic acid-microsomal system for testing in vitro possible oncogenic substances in animals and humans.     

Thermodynamic stability of bovine α-crystallin in its interactions with other bovine crystallins

Light scattering measurements were performed on dilute solutions of α-crystallin mixed with different combinations of β_H, β_L and γ-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of α-crystallin is progressively enhanced by its interactions with γ(β_H+γ)(β_H+β_L+γ) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.     

甘薯根腐病菌侵染对甘薯内源激素水平的影响

甘薯根腐病病原菌[Fusarium solani(Mart.)Sacc.f.sp.batatas McClure,简称FSB]侵染及其培养液滤液处理高敏感性甘薯品种‘胜利百号’后,引起甘薯叶片、茎尖和根部组织内源ABA含量大幅升高。其中在根部出现最早,但茎尖中积累浓度最高。侵染后甘薯叶片、茎尖和根部组织内源GA1/3含量显著低于对照。甘薯组培苗经FSB培养滤液处理9h后,ABA含量显著上升,处理15h,ABA含量呈下降趋势,而GA1/3含量在101和102稀释液处理15h(103稀释液处理12h)时出现显著上升。这些结果有助于解释甘薯根腐病株矮小不产生藤蔓,并在秋季大量现蕾开花的生理现象。     

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk

Excitotoxicity mediated via the ( S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l -glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨ_m), early release of cytochrome c (cyt c ), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of ΔΨ_m, and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke ΔΨ_m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c , SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.     

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg^− 1 of vitamin C (ascorbic acid or AA) and vitamin E (α-tocopherol or α-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p < 0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg^− 1 and on the hemoglobin concentration on treatment with 500 mg α-T kg^− 1 relatively to control. Mean corpuscular volume presented a significant decrease (p < 0.05) on treatment with 800 and 1200 mg AA kg⁻¹ when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p < 0.05) on treatment with 500 mg α-T kg^− 1. Only in vitamin C treatments, we noticed a significant increase (p < 0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg^− 1, had high total protein values compared to control. Fish treated with 800 or 1200 mg α-T kg^− 1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg^− 1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the permanently charged 1-methyl-4-phenylpyridinium species MPP(+), a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP(+) is thought to exert its toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-dependent accumulation of MPP(+) relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP resulted in 65-73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in the brain in relatively high concentrations. Unlike MPP(+), however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.     

Analysis of Rev1p and Pol zeta in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance

Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol zeta can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol zeta suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol zeta may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.