Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.     

Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.     

ER-60 domains responsible for interaction with calnexin and calreticulin

ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT). In this study, we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule. ER-60 was shown to be composed of at least four domains, named a, b, b', and a', by limited proteolysis. Recombinant fragments of ER-60, a, b', and a'c, were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient. These fragments each gave the circular dichroism (CD) spectrum of the folded protein. On the other hand, fragment b, which did not undergo the cooperative unfolding transition along a urea gradient gel, did not show any sign of the folded structure on the CD measurement. However, subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb'. Both a and a'c, which have a catalytic center CGHC motif, showed activity almost equivalent to half of that of wild-type ER-60. Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity, suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60. The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.     

Origin of magnetosome membrane: proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane

Prokaryotes are known to have evolved one or more unique organelles. Although several hypotheses have been proposed concerning the biogenesis of these intracellular components, the majority of these proposals remains unclear. Magnetotactic bacteria synthesize intracellular magnetosomes that are enclosed by lipid bilayer membranes. From the identification and characterization of several surface and transmembrane magnetosome proteins, we have postulated that magnetosomes are derived from the cytoplasmic membrane (CM). To confirm this hypothesis, a comparative proteomic analysis of the magnetosome membrane (MM) and CM of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1, was undertaken. Based on the whole genome sequence of M. magneticum AMB-1, 78 identified MM proteins were also found to be prevalent in the CM, several of which are related to magnetosome biosynthesis, such as Mms13, which is tightly bound on the magnetite surface. Fatty acid analysis was also conducted, and showed a striking similarity between the CM and MM profiles. These results suggest that the MM is derived from the CM.     

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 μmol photons · m^?2 · s^?1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700 μmol photons ? m^?2 ? s^?1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.