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The article discusses the result of the study regarding the psychrophilic and thermolabile malate dehydrogenase (TMD) of a novel Antarctic psychrotolerant, Flavobacterium frigidimaris, in Japan. The psychrophilic and TMD were purified to homogeneity from F. frigidimaris. The enzyme was susceptible to heat treatment and exhibits pro-R stereospecificity for hydrogen transfer. Compared to other malate dehydrogenases, the enzyme contained a number of amino acid residues and lower numbers of proline and arginine residues.

Previously, we reported that p38, which belongs to the mitogen-activated protein kinase (MAPK) superfamily, has an important role in the induction of apoptosis of cultured cerebellar granule neurons. However, the molecular mechanisms upstream of p38 activation remain unclear. Apoptosis signal-regulating kinase-1 (ASK1), a MAPK kinase kinase (MAPKKK) protein, is known to activate both c-Jun N-terminal kinase (JNK) and p38 via MAPK kinase (MKK) 4/7 and MKK3/6, respectively. Here, we examined whether ASK1 is involved in the activation of p38 in the low potassium (LK)-induced apoptosis of cerebellar granule neurons. We found that ASK1 was activated after a change to LK medium. In addition, the expression of ASK1-KM, a dominant-negative form of ASK1, using an adenovirus system was found to inhibit the activation of p38 and c-Jun and to prevent apoptosis. On the other hand, the expression of ASK1-ΔN, a constitutively active form of ASK1, activated p38 and c-Jun, but not JNK, another possible downstream target of ASK1. Furthermore, we examined the relationship between phosphatidylinositol 3-kinase (PI3-K) and ASK1. The addition of LY294002, a specific inhibitor of PI3-K, enhanced the ASK1 activity. These results indicate that ASK1 works downstream of PI3-K to regulate the p38–c-Jun pathway and apoptosis in cultured cerebellar granule neurons. [ABSTRACT FROM PUBLISHER]

Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphoryla-tion-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB. Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2β (FRS2β), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2β/ SNT2. Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellu-lar signaling pathways stimulated by TrkB receptor tyrosine kinase. [ABSTRACT FROM AUTHOR]

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherlchia co/i cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. 0. (1985) Proc. Natl Acad. Sci. USA 82, 4587 -4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coil as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp Bell fragment of &phis;105 DNA, and was released in the presence of 0.3 M KCI. [ABSTRACT FROM AUTHOR]

We obtained two PC12h cell lines, PC-pAB1 and PC-pAB2, stably expressing TrkB receptor and investigated the effects of BDNF and NT-3 in these cell lines. The cells differentiated into neuron-like cells in response to BDNF as well as NGF, neurite extension being more rapid in the former case. These TrkB-expressing cells also extended neurites in response to NT-3, which is a nonpreferred ligand of TrkB. Next, we examined the survival-promoting effects of NGF, BDNF, and NT-3 under apoptotic conditions of oxygen toxicity in naive cells and NGF deprivation in differentiated cells. In both cases, BDNF prevented cell death similarly to NGF. NT-3 prevented cell death induced by oxygen toxicity in naive cells, but not that induced by NGF deprivation in differentiated cells. NT-3 induced the tyrosine phosphorylation of TrkB in naive cells, but not in differentiated cells. These results indicate that NT-3 has survival-promoting effects on naive TrkB-expressing PC12h cells, but not on differentiated cells because of its inability to induce the tyrosine phosphorylation of TrkB. [ABSTRACT FROM AUTHOR]

The article presents an abstract of the study "BDNF Stimulates Interaction of SHP2 With PI 3-K and GRB2 in Cultured Cerebral Cortical Neurons." The study will be presented at a joint meeting of the International Society for Neurochemistry and the European Society for Neurochemistry which will be organized in Berlin, Germany between August 8 to 14, 1999.

On cell maturation following culture in medium containing 26 mM potassium (high K+; HK), a change to medium containing 5 mM potassium (low K+; LK) rapidly induces apoptosis in rat cerebellar granule neurons. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) have survival-promoting effects on the neurons via PI3-K. However, it remains unclear how they prevent the apoptosis in the pathway downstream of phosphatidylinositol-3 kinase (PI3-K). Recently, we have reported that PI3-K-ASK1 pathway is involved in signal-transduction to p38 MAPK (p38)-c-Jun pathway. Here we found that IGF-1 had a greater survival-promoting effect than BDNF, and activated PI3-K to a higher level and maintained the level for a longer time. BDNF and IGF-1 suppressed the activation of p38 and c-Jun, but not of c-Jun N-terminal kinase (JNK), caused by lowering the potassium concentration. The inhibitory effects of IGF-1 were much greater than those of BDNF. In addition, LY294002, a specific inhibitor of PI3-K, cancelled the inhibitory effects of BDNF and IGF-1. These results suggest that the greater inhibitory effects of IGF-1 than BDNF, on activation of p38 and c-Jun and apoptosis, are caused by the higher level of PI3-K activation during LK-induced apoptosis of cultured cerebellar granule neurons. [Copyright &y& Elsevier]