Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Cretaceous coleopteran larva fed on a female fructification of extinct gymnosperm

A fossil coleopteran larva was found in a permineralized female fructification of a new extinct gymnosperm from the Late Cretaceous of Japan. The fossil provides the firstin situ evidence that the insects used some gymnosperm fructification as foods as well as pupation sites before they started using angiosperm fruits.     

Interannual fluctuations in recruitment and growth of the sardine,Sardinops melanostictus,in the sea of Japan and adjacent waters

Recruitment and growth of the sardineSardinops melanostictus fluctuated markedly in the Sea of Japan and adjacent waters between 1978 and 1993. Stock size was calculated using Virtual Population Analysis and average body length in each age class was determined by the number of annual rings on the scales. There is an inverse correlation between average water temperature at a depth of 50 m in the coastal area of the mainland of Japan in winter (January to March) and recruitmentR defined as the number of individuals at 1 year old. There is also an inverse correlation between spawning stock sizeE and reproductive success in (R/E). A multiple regression model using spawning stock size and water temperature in winter as independent variables can explain 73% of variance in reproductive success. It suggests that both density-dependent and density-independent factors perform important roles determining reproductive success. There is an inverse correlation between body length and stock size and this suggests that there is a density-dependent effect on the growth of the sardine.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Archiascomycetes: detection of a major new lineage within the Ascomycota

For phylogenetic analysis of the higher fungi, we sequenced the nuclear small subunit rRNA (18S rRNA) gene fromTaphrina populina, the type species of the genusTaphrina, andProtomyces lactucae-debilis. The molecular phylogeny inferred from these 2 sequences and 75 sequences from the DNA data bank divided the Ascomycota into three major lineages: the hemiascomycetes, the euascomycetes, and the archiascomycetes, newly described herein. The former two lineages are monophyletic, whereas the archiascomycetes, which originated first and are comprised ofTaphrina, Protomyces, Saitoella, Schizosaccharomyces, andPneumocystis, may not be monophyletic. Among the archiascomycetes, theTaphrina/Protomyces branch is monophyletic. Confirmation of the archiascomycetes as a monophyletic taxonomic class will require comparison of additional genetically defined characters.This work was supported in part by grants 05454030 from the Ministry of Education, Science, and Culture of Japan (to J. S.) and 4369 from the Japan Society for the Promotion of Science Fellowship Programs (to H. N.).