Atorvastatin-induced cardioprotection is mediated by increasing inducible nitric oxide synthase and consequent S-nitrosylation of cyclooxygenase-2

We determined the effects of cyclooxygenase-1 (COX-1; SC-560), COX-2 (SC-58125), and inducible nitric oxide synthase (iNOS; 1400W) inhibitors on atorvastatin (ATV)-induced myocardial protection and whether iNOS mediates the ATV-induced increases in COX-2. Sprague-Dawley rats received 10 mg ATV.kg(-1).day(-1) added to drinking water or water alone for 3 days and received intravenous SC-58125, SC-560, 1400W, or vehicle alone. Anesthesia was induced with ketamine and xylazine and maintained with isoflurane. Fifteen minutes after intravenous injection rats underwent 30-min myocardial ischemia followed by 4-h reperfusion [infarct size (IS) protocol], or the hearts were explanted for biochemical analysis and immunoblotting. Left ventricular weight and area at risk (AR) were comparable among groups. ATV reduced IS to 12.7% (SD 3.1) of AR, a reduction of 64% vs. 35.1% (SD 7.6) in the sham-treated group (P < 0.001). SC-58125 and 1400W attenuated the protective effect without affecting IS in the non-ATV-treated rats. ATV increased calcium-independent NOS (iNOS) [11.9 (SD 0.8) vs. 3.9 (SD 0.1) x 1,000 counts/min; P < 0.001] and COX-2 [46.7 (SD 1.1) vs. 6.5 (SD 1.4) pg/ml of 6-keto-PGF(1alpha); P < 0.001] activity. Both SC-58125 and 1400W attenuated this increase. SC-58125 did not affect iNOS activity, whereas 1400W blocked iNOS activity. COX-2 was S-nitrosylated in ATV-treated but not sham-treated rats or rats pretreated with 1400W. COX-2 immunoprecipitated with iNOS but not with endothelial nitric oxide synthase. We conclude that ATV reduced IS by increasing the activity of iNOS and COX-2, iNOS is upstream to COX-2, and iNOS activates COX-2 by S-nitrosylation. These results are consistent with the hypothesis that preconditioning effects are mediated via PG.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Murine alpha-fetoprotein: N-terminal amino acid sequence and C-terminal residue.

We have purified to homogeneity murine alpha-fetoprotein (MAFP) and determined the amino acid sequence of the first twenty-four residues. The N-terminal sequence obtained shows a high degree of homology with human and rat AFP's, but not human or rat albumins. The C-terminal residue is the same as human and “slow” rat AFP, but different from the corresponding albumins. We conclude that the AFP's are derived from homologous genes which are at best distantly related to the ancestral gene for albumin. The single C-terminal residue and N-terminal sequence suggests that the multiple forms of MAFP observed by others are due to carbohydrate micro-heterogeneity.     

UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.     

The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase

Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²⁺] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.