Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.     

Structure-function relationship of bromelain isoinhibitors from pineapple stem

Bromelain isoinhibitors from pineapple stem (BIs) are unique double-chain inhibitors and inhibit the cysteine proteinase bromelain competitively. The three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded anti-parallel beta-sheet. Unexpectedly, BIs were found to share similar folding and disulfide-bond connectivities not with the cystatin superfamily, but with Bowman-Birk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.     

Determination of the NMR structure of Gln25-ribonuclease T1

Ribonuclease (RNase) T1 is a guanyloribonuclease, having two isozymes in nature, Gln25- and Lys25-RNase T1. Between these two isozymes, there is no difference in catalytic activity and three-dimensional structure; however, Lys25-RNase T1 is slightly more stable than Gln25-RNase T1. Recently, it has been suggested that the existence of a salt bridge between Lys25 and Asp29/Glu31 in Lys25-RNase T1 contributes to the stability. To elucidate the effects of the replacement of Lys25 with a Gln on the conformation and microenvironments of RNase T1 in detail, the three-dimensional solution structure of Gln25-RNase T1 was determined by simulated-annealing calculations. As a result, the topology of the overall folding was shown to be very similar to that of the Lys25-isozyme except for some differences. In particular, there were two differences in the property of torsion angles of the two disulfide bonds and the conformations of the residues 11-13, 63-66, and 92-93. With regard to the residues 11-13, the lack of the above-mentioned salt bridge in Gln25-RNase T1 was thought to induce the conformational difference of this segment as compared with the Lys25-isozyme. Furthermore, it was proposed that the perturbation of this segment might transfer to the residues 92-93 via the two disulfide bonds.     

Src homology region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.     

Recovery of Bacillus thuringiensis from Marine Sediments of Japan

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Received: 29 November 1999 / Accepted: 10 January 2000     

Strongyloides venezuelensis: heparin-binding adhesion substances in immunologically damaged adult worms

Immunologically damaged Strongyloides venezuelensis adult worms were examined for their mucosal invasion ability and secretion of heparin-binding adhesion substances. S. venezuelensis was expelled from male Wistar rats 4 to 5 weeks after infection. Four-week-old adult worms were smaller and had fewer eggs than 1-week-old adult worms. One-week-old, 4-week-old, and 5-week-old adult worms equally established in the recipient mouse intestine when surgically implanted. Adult worms of 4 and 5 weeks of age secreted adhesion substances as much as 1-week-old adult worms. There was no difference in the heparin-binding activities and the lectin-binding profile of adhesion substances among adult worms of different ages. The rate of secretion of adhesion substances from the mouth was also identical. Heparin-binding activities were detected in crude adult worm proteins; however, proteins of 5-week-old adult worms had weaker heparin-binding activities than those of 1-week-old adult worms. Western blotting revealed that a number of heparin-binding proteins were lost in 5-week-old adult worms. A heparin-binding protein of 42. 0 kDa, which was consistently expressed in adult worms, was a possible component of heparin-binding adhesion substances which are secreted from the mouth.     

Correlation of oxidative transformations of hydrolyzable tannins and plant evolution

The progressive oxidative transformations in the biogenesis of hydrolyzable tannins, gallotannin (I) --> ellagitannin (II) --> dehydro ellagitannin (III) --> transformed dehydroellagitannin (IV), conform in several aspects to the evolutionary routes in the subclasses of Dicotyledonae, particularly in the Rosidae.     

Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.     

1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.