Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.     

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.     

Neovascularization with blood-brain barrier breakdown in delayed neuronal death

Various kinds of acute pathological events in the central nervous system, such as ischemia, hemorrhage, and trauma, often cause brain edema. The edema may advance for days or weeks while inducing extensive damage in neural function, regardless of the extent of the original damage, and often results in death. Delayed edema is thought to be vasogenic; however, the mechanism underlying edema induction remains unknown. We found delayed vascular cell proliferation with a blood-brain barrier breakdown in and around the gerbil CA1 hippocampus, a region known to be involved in delayed apoptotic neuronal death 2-6 days after transient ischemia. Vascular cell proliferation, assessed by (3)H-thymidine incorporation, was most prominent 4-6 days after ischemia, and extravasation of exogenously applied dye or endogenous serum albumin from blood vessels was observed concomitantly. We propose neovascularization in delayed neuronal death as a cause of brain edema advancing days after neurological events.     

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of d-3-hydroxyacyl-CoA dehydratase/d-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.     

Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library.

Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

The BMP-related protein radar: a maintenance factor for dorsal neuroectoderm cells?

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.     

A decrease in temperature triggers a luminescent response in Noctiluca scintillans

Noctiluca scintillans (Macartney) Kofoid, a typical bioluminescentorganism, is known to respond to a mechanical stimulus by emittinglight. We found that a luminescent response could also be triggeredby lowering the temperature of the environment, but not by raisingit. This response was analyzed with respect to the rate of decreasein temperature. The results showed that the probability of theresponse was positively related to the rate of the decreasein temperature. The sites of initiation of the luminescent responsewere distributed randomly all over the surface of the cell body.The flash response that was triggered by a decrease in temperaturewasquite similar to that triggered by a mechanical stimulusat the lower temperature in terms of duration, as wellas interms of the velocity of expansion of the excited region. Afatal response, namely a slowly propagating flash (SPF), wasalso recognized.     

Simultaneous determination of noradrenaline and 3-methoxy-4-hydroxyphenylglycol in plasma with high-performance liquid chromatography using electrochemical detection

We herein report the simultaneous determination of the levels of noradrenaline (NA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), a major metabolite of NA. The sample was subjected to a Sep Pak C18 cartridge prior to the NA and MHPG assay by high-performance liquid chromatography with an electrochemical detector. The results correlated well with the established methods. The average percentage of recovery was 91.2 and 98.7% for NA and MHPG, respectively. The intraassay coefficients of variation were 3.7 and 4.6% for NA and MHPG. The interassay coefficients of variation were 3.5 and 7.5% for NA and MHPG, respectively.