Grafting neural stem cells improved the impaired spatial recognition in ischemic rats.

To determine the possible therapeutic potential of neural stem cells (NSCs) introduced into the damaged central nervous system, we grafted adult hippocampus-derived NSCs into the hippocampus of rats with transient global ischemia. Transient four-vessel occlusion yielded 90-95% losses of pyramidal neurons in the hippocampal CA1 region. In this region, 1-3% of the grafted cells survived; and 3-9% of them expressed NeuN, a neuronal marker. Rats with more than 120 NeuN-positive cells showed partial improvement of impaired spatial learning in a water maze test. These results suggest that NSCs grafted in the ischemic brain are able to differentiate into neurons and to improve spatial recognition.

Haemopoietic biglycan produced by brain cells stimulates growth of microglial cells.

We have recently found that soluble biglycan purified from rat thymic myoid cells had haemopoietic activity capable of inducing preferential growth and differentiation of monocytic lineage cells from various haemopoietic sources, including brain microglial cells. In the present study, to understand developmental mechanisms of microglial/monocytic cells in the brain, we have attempted to identify haemopoietic activity of the brain biglycan. The mRNA and the immunological epitope of biglycan were detected in the rat brain homogenates and several rat glial cell lines. Immunohistochemical study showed that several different types of brain cells produced biglycan. During development biglycan synthesis in the brain appeared to be increased. The brain haemopoietic biglycan was easily separated by DEAE-Sepharose chromatography from the macrophage colony stimulating factor (M-CSF) which was concomitantly produced from the brain cells. The brain haemopoietic biglycan, purified through immunoaffinity column, indeed stimulated growth of primarily cultured microglial cells. Taken together, these results suggest that the haemopoietic biglycan plays an important role in generating brain-specific circumstances for development of microglial/monocytic cells.

JNK activation is associated with intracellular beta-amyloid accumulation.

c-Jun has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to c-Jun activation in AD is not known. Activation of c-Jun N-terminal kinase (JNK) is obviously a candidate for the upstream event. We tested this possibility focusing on PS1-linked AD. First, we observed that JNK is actually activated in cerebral neurons of PS1-linked AD patients, using immunohistochemistry and Western blot analyses with anti-activated JNK antibodies. We analyzed the relationship between beta-amyloid (beta A) and JNK activation by using aged transgenic mice overexpressing mutant (M146L) PS1 and human AD brains. The mice showed no neuronal loss but a very few diffuse beta A deposits, corresponding to the early stage of PS1-linked AD brain. Some neurons were reactive for anti-beta A antibodies in the cerebral cortex. Interestingly, JNK activation was observed in neurons showing intracellular beta A immunoreactivity in transgenic mice. Association between intracellular beta A and JNK activation was confirmed in cortical neurons of sporadic and PS1-linked AD patients. Furthermore, introduction of beta A peptides into the primary culture cortical neurons induced JNK activation and cell death. Collectively, these results suggested that intracellular beta A accumulation might trigger JNK activation leading to neuronal death.

Analysis of lymphoproliferative cytokines produced by thymic myoid cells.

Lymphoproliferative activities produced by cloned thymic myoid cell 871207B were analysed by immunological and biochemical methods. The lymphoproliferative activities were separated into two fractions by DEAE-Sepharose CL-6B chromatography: one is in the fraction passed through the column and the other in the fraction eluated from the column with a low concentration of NaCl. The eluated fraction induced the proliferation of interleukin-1 (IL-1)-dependent D10N4 M cells. This activity was abrogated by an anti-IL-1 alpha antibody, but not an anti-IL-1 beta antibody. Expression of IL-1 alpha mRNA was also detected in 871207B cells. The thymocyte proliferative activity found in the fraction passed through the DEAE-Sepharose column was further separated into three fractions by heparin-Sepharose column chromatography: (1) the fraction passed through the column, (2) the fraction weakly bound to the column, and (3) the fraction firmly bound to the heparin column. The fraction passed through the heparin column sustained the growth of IL-6-dependent MH60.BSF-2 cells. IL-6-specific mRNA was found in 871207B cells. The thymocyte proliferative activity of the fraction firmly bound to the heparin column was neutralized with an anti-IL-7 antibody. The biological activity of the fraction weakly bound to the column remained to be elucidated. These results suggest that thymic myoid cells produce IL-1 alpha, IL-6, IL-7 and unidentified lympho-stimulatory factors, all of which play significant roles in many steps of T-cell development in the thymus.

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds.

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant beta-tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10-20 transformants per micrograms of DNA per 4-8 x 10(7) viable protoplasts. Integration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems in the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.

Immunological and biochemical characterization of biglycan-like haemopoietic factor.

Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L-929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N-glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells.

Action of a nuclease from rat nuclei on UV-irradiated DNA.

An Mg(2+)-dependent nuclease was highly purified from rat-liver nuclei. The nuclease activity was enhanced in ultraviolet light (UV)-irradiated dsDNA, but not in unirradiated dsDNA. Irrespective of UV irradiation, ssDNA was readily cleaved by this enzyme. UV-irradiated plasmid DNA was incubated with this enzyme and subjected to the template primer activity assay with a Klenow polymerase. With increasing incubation time, the activity was enhanced in the circular-relaxed and linear forms, but not in the superhelical form. These results implied that this enzyme excises UV-damaged sites in dsDNA to form single strand gaps for repair synthesis.

Vasorelaxant and hypotensive effects of tilisolol hydrochloride (N-696) in isolated rat thoracic aorta and pithed rats.

Vasorelaxant and hypotensive effects of tilisolol hydrochloride (N-696) in isolated rat thoracic aorta and pithed rats were investigated. In rat thoracic aorta pre-contracted with KCl (20 mM), tilisolol (10(-5)-10(-3) M) produced concentration-related relaxation, but nadolol and atenolol did not significantly inhibit the responses to 20 mM KCl. The concentration-relaxation curve of tilisolol underwent rightward parallel shifts only to a limited extent in the presence of glibenclamide, a specific antagonist of K+ channel openers. Glibenclamide also shifted the concentration-relaxation curve of cromakalim to the right and in a parallel manner, whereas it did not change that of propranolol. In pithed rats, tilisolol (0.5-2.0 mg/kg, i.v.), but neither nadolol nor atenolol, caused a dose-dependent decrease in diastolic blood pressure and a slight increase in heart rate. Following treatment of the preparation with glibenclamide, the hypotensive effects of tilisolol and cromakalim were antagonized, while that of propranolol was not affected. These results suggest that the vasorelaxant and hypotensive actions of tilisolol involve an opening of K+ channels which can be inhibited by glibenclamide and may also involve additional relaxant mechanisms of action independent of K+ channel opening.

Bending of a highly repetitive component in rat nuclear DNA.

A highly repetitive component in rat nuclear DNA was isolated by HindIII digestion and cloned. A 370-bp cloned component was highly AT-rich (68.3%) in about one third of the region from the 3'-terminus and showed an anomalously slow gel electrophoretic mobility (k-factor = 1.19). These results indicated that a sequence-directed bending of the helix axis occurs in the component. Accordingly, a subclone containing a tandem dimer of the component was isolated and subjected to a circular permutation analysis for exploring the bend center (1). In consequence, the center was shown to be present in the sequence ranging from position near 270 to the 3'-terminus and estimated to be located around position 340.

A nuclease from rat-liver nuclei with endo- and exonucleolytic activity.

An Mg2(+)-dependent endonuclease endogenous to rat-liver nuclei had an exonuclease activity for single-stranded DNA, but not for duplex DNA. The activity was about twice as high in the 3'----5' direction as in the 5'----3' direction. The products by 3'----5' activity were mononucleotides alone. The 5'----3' activity released mononucleotides as main products and small amounts of di-, tri-, tetra- and oligonucleotides. Another major endonuclease endogenous to the nuclei, a Ca2+/Mg2(+)-dependent endonuclease, did not have such exonuclease activities.

Base sequences of highly repetitive components in nuclear DNAs from rat liver and rat-ascites hepatoma.

A 370-bp highly repetitive component in each of the nuclear DNAs from rat liver (RL) and rat-ascites hepatoma (AH) was isolated by HindIII digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced. Heterogeneity of size was found in 7 of the RL clones (366-369 bp), but in only 2 of the AH clones (369 bp). The sequence homology was 64.6% among the RL clones; 80.3% among the AH clones. The base compositions were AT-rich, ranging from 61.1% to 64.7%. Many A and/or T runs consisting of 2-5 bases were interspersed throughout each sequence. The restriction sites reported previously, EcoRI, HaeIII, HindIII, HinfI and HphI sites, were confirmed in almost all of the clones. In the present experiment, 12 kinds of the sites were further found in both RL and AH clones.

Affinity of a DNA with highly repetitive sequence for nuclear proteins from rat liver.

A nuclear scaffold fraction (designated P fraction elsewhere) comparable to a nuclear matrix was prepared from rat liver. This fraction was composed mainly of 45-49 kDa proteins and high-molecular-weight proteins (more than 90 kDa). In addition, a 370-bp repetitive sequence DNA fragment was derived predominantly from the EcoRI digest of the deproteinized P fraction. By an immunoblot affinity assay with the P fraction, the fragment was shown to have affinity for each of the 107- and 115-kDa proteins. Moreover, by a filter binding assay with a mixture of these proteins, the affinity level was estimated to be about 6 times as high in the native (double-stranded) fragment as in the denatured (single-stranded) fragment.