Spinal heat shock protein (70) expression: effect of spinal ischemia,hyperthermia (42 degrees C)/hypothermia (27 degrees C), NMDA receptor activation and potassium evoked depolarization on the induction

The present study shows that anoxic neuronal depolarization or NMDA receptor activation are potent stimuli for inducing spinal neuronal heat shock protein 70 (Hsp70). Spinal hyperthermia, despite its significant glutamate releasing effect, induced only glial Hsp70 upregulation. No significant increase in spinal Hsp70 expression after potassium depolarization was seen. Transient spinal ischemia (6 min) was induced by the inflation of a 2F Fogarty catheter placed into descending thoracic aorta during concurrent hypotension (40 mmHg). To determine the onset of anoxic depolarization extracellular concentration of K+ was measured in the lumbar dorsal horn using a microelectrode. Spinal hyperthermia (42 degrees C) or hypothermia (27 degrees C) was induced using a heat exchanger placed in the paravertebral subcutaneous space overlying Th5-S4 spinal segments. To measure extracellular concentration of glutamate during hyperthermia a loop dialysis catheter was implanted into lumbar intrathecal space. Receptor specific (NMDA, 3 microg) or non-specific (KCl, 10 microl, 1M) neuronal depolarization was induced using previously implanted intrathecal catheters. After ischemia, temperature manipulations or drug injections animals survived for 4 or 24h. Animals were then terminally anesthetized and perfusion fixed for Hsp70 immunohistochemistry. After spinal ischemia or NMDA administration a neuronal Hsp70 expression was seen at 24h. After spinal hyperthermia only glial expression was seen at 4h. Hyperthermia significantly increased CSF glutamate concentration, however, MK-801 (a non-competitive NMDA receptor antagonist) pretreatment failed to block Hsp70 expression. After hypothermia or potassium depolarization only minimal or no Hsp70 expression was seen in glial cells. Exposure of neuronal tissue to a specific stimuli may lead to intervals of increased resistance to subsequent neurotoxic/ischemic insult. The intervening biochemistry of this protection has been attributed to a family of molecules referred to as HSP. In the present study, we demonstrate that short-lasting anoxic depolarization or activation of NMDA receptor are the most potent stimuli for spinal neuronal Hsp70 induction. This effect corresponds with the observed ischemic tolerance state induced by short-lasting preconditioning spinal ischemia.     

Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat. In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER. Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed. After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER. The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector. Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration. No inhibitory effects were observed 10 and 14 days after AxASPA administration. In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER. Survival rates did not differ between AxASPA- and AxLacZ-treated SERs. Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.     

Three-dimensional structural views of branch migration and resolution in DNA homologous recombination

The processing of the Holliday junction by various proteins is a major event in DNA homologous recombination and is crucial to the maintenance of genome stability and biological diversity. The proteins RuvA, RuvB and RuvC play central roles in the late stage of recombination in prokaryotes. Recent atomic views of these proteins, including protein-protein and protein-junction DNA complexes, provide new insights into branch migration mechanisms: RuvA is likely to be responsible for base-pair rearrangements, whereas RuvB, classified as a member of the AAA(+) family, functions as a pump to pull DNA duplex arms without segmental unwinding. The mechanism of junction resolution by RuvC in the RuvABC resolvasome remains to be elucidated.     

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone

A Wilms tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a prophylactic model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a therapeutic setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a therapeutic model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.H. Nakajima and K. Kawasaki contributed equally to this study.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk

When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.     

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.     

L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.