GIT1 mediates Src-dependent activation of phospholipase Cgamma by angiotensin II and epidermal growth factor

Critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase Cgamma (PLCgamma) and elevation of intracellular calcium. c-Src has been proposed as a common mediator for these signals activated by both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein-1 (GIT1) is a substrate for c-Src that undergoes tyrosine phosphorylation in response to angiotensin II (AngII) and EGF in vascular smooth muscle and 293 cells. GIT1 associates with PLCgamma via the PLCgamma Src homology 2 and 3 domains constitutively, and the interaction is unaltered by AngII and EGF. GIT1 interaction with PLCgamma is required for PLCgamma activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides. GIT1 interacts with PLCgamma via a novel Spa homology domain (SHD) and a coiled-coil domain. Deletion mutation analysis showed that GIT1(SHD) is required for AngII- and EGF-mediated PLCgamma activation (measured by phosphorylation of Tyr783 and inositol 1,4,5-trisphosphate formation). We propose that GIT1 is a novel regulator of PLCgamma function that mediates PLCgamma activation by c-Src and integrates signal transduction by GPCRs and TKRs.     

Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease

Cerebral amyloid angiopathy (CAA) due to beta-amyloid (Abeta) is one of the specific pathological features of familial Alzheimer's disease. Abeta mainly consisting of 40- and 42-mer peptides (Abeta40 and Abeta42) exhibits neurotoxicity and aggregative abilities. All of the variants of Abeta40 and Abeta42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the Abeta40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type Abeta40. Similar tendency was observed for Abeta42 mutants at positions 22 and 23 whose neurotoxicity was 50-200 times stronger than that of the corresponding Abeta40 mutants, suggesting that these Abeta42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-Abeta42 and D23N-Abeta42 was similar to that of wild-type Abeta42, E22Q-Abeta42 and E22K-Abeta42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of Abeta mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that beta-sheet content of the Abeta mutants correlates with their aggregation. However, beta-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue beta-turn significantly enhanced the aggregative ability.     

Functional inhibition of the p75 receptor using a small interfering RNA

The neurotrophin receptor p75(NTR) mediates a wide variety of biological effects. Consistent with the function in controlling the survival and neurite formation, p75(NTR) is expressed during the developmental stages of the nervous system. Importantly, p75(NTR) is re-expressed in various pathological conditions and is suggested to contribute to the inhibition of neuronal regeneration and the death of the neurons. Here we develop a tool to knock down the expression of p75(NTR) by employing a small interfering RNA (siRNA). The siRNA for p75(NTR) effectively reduces the expression of endogenous p75(NTR) both in Schwann cells and dorsal root ganglion neurons in vitro. NGF-induced cell death in Schwann cells and the neurite retraction in DRG neurons induced by myelin-associated glycoprotein are attenuated by the siRNA. Inhibition of p75(NTR) in specific pathological conditions by the siRNA may provide a potential therapeutic agent.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Maximization of hydrogen production ability in high-density suspension of Rhodovulum sulfidophilum cells using intracellular poly(3-hydroxybutyrate) as sole substrate

Growth of and hydrogen production by wild-type (WT) Rhodovulum sulfidophilum were compared with those by one of its mutants lacking the poly(3-hydroxybutyrate) (PHB) biosynthesis ability (PNM2). During phototrophic growth under aerobic conditions with fixed illumination, changes in the extinction coefficient and PHB content of WT and PNM2 cells revealed interference of light penetration by PHB. WT cells synthesized PHB at an early stage of the cultivation. PHB degradation after exhaustion of acetate during the cultivation of WT resulted in a decrease of the extinction coefficient. The hydrogen production rate under anaerobic conditions with fixed illumination was examined in WT and PNM2 cell suspensions at different densities. The hydrogen production rate was determined not by the light penetration but by the kinds of hydrogen donors and the density of suspension. The highest value of the rate of hydrogen production from PHB, 33.0 ml/l/h, was improved compared with 26.6 ml/l/h, which was the highest value in hydrogen production from succinate. Under the same illumination, conversion to hydrogen from PHB is more efficient than that from succinate, which is one of the best substrates for hydrogen production. These results suggest that the hydrogen production rate can be maximized in the hydrogen production system based on PHB degradation, which is achieved in high-density suspension under external-substrate-depleted conditions after aerobic cultivation in the presence of an excess amount of acetate.     

Cloning and expression of the superoxide dismutase gene from the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F)

We identified the SOD gene in the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F) and constructed a high-level expression system in Escherichia coli. A 2.6-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SmaI contained the SOD gene and part of another open reading frame. The amino acid sequence deduced from the SOD gene, which was composed of 238 amino acid residues, showed high homogeneity with iron-containing SOD (Fe-SOD) and predicted that the amino terminus of this protein would carry an export signal peptide. We produced the precursor form of SOD (PSOD) and the mature form of SOD (MSOD), which lacked the putative signal peptide. In E. coli, PSOD was present in insoluble inclusion bodies, and its putative signal peptide was not cleaved. In contrast, MSOD contained one iron per mononer and formed a dimer, which exhibited an SOD activity of 850 U/mg. Furthermore, D. vulgaris soluble extract showed a band of SOD activity on native polyacrylamide gel that migrated to the same point as MSOD. The intracellular localization of SOD and its role in D. vulgaris are also discussed.     

New allyl ester linker and solid-phase block synthesis of the serglycin core region

The prototype glycopeptidyl fragments of serglycin, a proteoglycan with the characteristic peptide sequence of repeating L-seryl-L-glycine, were synthesized by a convergent method involving block condensation on a solid support. In order to facilitate detachment of the protected glycopeptides from the resin, a new allyl ester type of linker, which is cleavable by Pd(O)-catalysis, was designed and used in combination with the commercial acid-labile Sieber amide resin for the solid-phase synthesis. Glycopeptide blocks consisting of [O-(2,3,4-tri-O-acetyl-D-xylosyl)-L-seryl-L-glycine]n (n = 1 - 8) were produced in good yields. Block condensation in a solution was also successful to synthesize up to the hexadecapeptide (n = 8).     

Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post-injured sciatic nerve which was enriched in non-myelinating Schwann cells using cDNA microarrays. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down-regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co-receptor for glial cell line-derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.     

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [-247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1beta, and 15-deoxy Delta12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.