The Structure of Graphinone

A microorganism M–2 was isolated as a strain capable of converting (—)-menthone to other compounds. The strain was identified as Pseudomonas fluorescens by taxonomical investigation. The conversion products of (—)-menthone were determined to be (—)-t-4-isopropyl-3-oxo-r-l-cyclohexanecarboxylic acid,* (+)-c-4-isopropyl-3-oxo-r-1-cyclohexane-carboxylic acid* and (+)-t-3-hydroxy-t-4-isopropyI-r-l-cyclohexanecarboxylic acid.* As the main pathway, it was proposed that (—)-menthone was oxidized to a keto acid which was successively reduced to a hydroxy acid.     

Determination of L-Cysteine with L-Methionine γ-Lyase

A chemically defined medium was devised to examine the growth, production and biochemical pathway of tetrocarcin A. The production of tetrocarcin A was greatly stimulated by l-feucine and its corresponding keto acid, α-ketoisocaproate, suggesting that l-leucine is involved in the biosynthesis of tetrocarcin A. About 10–12 μg/ml of tetrocarcin A was produced in a chemically defined medium consisting of 20 g sucrose, 2.5 g KNO₃, 5 g MgSO₄·7H₂O, 5 g KH₂PO₄ and 1 g l-leucine per liter of water (pH 7.0).     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

Involvement of Ceramide in the Mechanism of Cr(VI)-induced Apoptosis of CHO Cells

Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.     

Mechanism of α-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of α-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca²⁺]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (～100 μM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.     

Blockade of the TP receptor attenuates the exercise pressor reflex in decerebrated rats with chronic femoral artery occlusion

Cyclooxygenase metabolites stimulate or sensitize group III and IV muscle afferents, which comprise the sensory arm of the exercise pressor reflex. The thromboxane (TP) receptor binds several of these metabolites, whose concentrations in the muscle interstitium are increased by exercise under freely perfused conditions and even more so under ischemic conditions, which occur in peripheral artery disease. We showed that the exercise pressor reflex is greater in rats with simulated peripheral artery disease than in rats with freely perfused limbs. These findings prompted us to test the hypothesis that the TP receptor contributes to the exaggerated exercise pressor reflex occurring in a rat model of peripheral artery disease. We compared the cardiovascular responses to static contraction and stretch before and after femoral arterial injections of daltroban (80 μg), a TP receptor antagonist. We performed these experiments in decerebrate rats whose femoral arteries were ligated 72 h before the experiment (a model of simulated peripheral artery disease) and in control rats whose hindlimbs were freely perfused. Daltroban reduced the pressor response to static contraction in both freely perfused (n = 6; before: Δ12 ± 2 mmHg, after: Δ6 ± 2 mmHg, P = 0.024) and 72-h-ligated rats (n = 10; before: Δ25 ± 3 mmHg, after: Δ7 ± 4 mmHg, P = 0.001). Likewise, daltroban reduced the pressor response to stretch in the freely perfused group (n = 9; before: Δ30 ± 3 mmHg, after: Δ17 ± 3 mmHg, P < 0.0001) and in the ligated group (n = 11; before: Δ37 ± 5 mmHg, after: Δ23 ± 3 mmHg, P = 0.016). Intravenous injections of daltroban had no effect on the pressor response to contraction. We conclude that the TP receptor contributes to the pressor responses evoked by contraction and stretch in both freely perfused rats and rats with simulated peripheral artery disease.     

Ran and Calcineurin Can Participate Collaboratively in the Regulation of Spermatogenesis in Scallop

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that plays important roles in the transduction of calcium signals in a variety of tissues. In addition, calcineurin has been implicated in the process of spermatogenesis. A novel calcineurin-binding protein, CaNBP75, has been identified in scallop testis. The C-terminal region of CaNBP75 is homologous to the C-terminal region of RanBP3, a Ran-binding domain-containing protein. A small G protein Ran has been involved in spermiogenesis by virtue of the fact that its localization in spermatids changes during spermiogenesis. The current study was performed to investigate the functions of Ran and CaNBP75 in the regulation of calcineurin in testis to further understand the basic functions of calcineurin during spermatogenesis. First, cloning and sequencing of a scallop Ran cDNA isolated from testis revealed that scallop Ran is well-conserved at the amino acid level. Secondly, direct binding of Ran to CaNBP75 was demonstrated in an in vitro pull-down assay. Thirdly, analysis of the tissue distribution of Ran, CaNBP75, and calcineurin showed that these proteins are abundantly expressed in testis. Fourthly, comparison of the expression profiles of Ran and CaNBP75 with that of calcineurin in scallop testis during the maturation cycle revealed that Ran and CaNBP75 mRNA levels increase during meiosis and spermiogenesis, similar to calcineurin. Finally, co-immunoprecipitation analysis suggests that Ran, CaNBP75, and calcineurin interact in scallop testis during maturation. These results suggest that Ran, CaNBP75, and calcineurin may act in a coordinated manner to regulate spermatogenesis.