Pharmacologic preconditioning effects: Prostaglandin E1 induces heat-shock proteins immediately after ischemia/reperfusion of the mouse liver

Prostaglandin E1 (PGE1) has several potential therapeutic effects, including cytoprotection, vasodilation, and inhibition of platelet aggregation. This study investigates the protective action of PGE1 against hepatic ischemia/reperfusion injury in vivo using a complementary DNA microarray. PGE1 or saline was continuously administered intravenously to mice in which the left lobe of the liver was made ischemic for 30 minutes and then reperfused. Livers were harvested 0, 10, and 30 minutes postreperfusion. Messenger RNA was extracted, and the samples were labeled with two different fluorescent dyes and hybridized to the RIKEN set of 18,816 full-length enriched mouse complementary DNA microarrays. Serum alanine aminotransferase and aspartate aminotransferase levels at 180 minutes postreperfusion were significantly lower in the PGE1-treated group than in the saline-treated group. The cDNA microarray analysis revealed that the genes encoding heat-shock protein (HSP) 70, glucose-regulated protein 78, HSP86, and glutathione S-transferase were upregulated at the end of the ischemic period (0 minutes postreperfusion) in the PGE1 group. Our results suggested that PGE1 induces HSPs immediately after ischemia reperfusion. HSPs might therefore play an important role in the protective effects of PGE1 against ischemia/reperfusion injury of the liver.     

IgG Anti-GM1 antibody is associated with reversible conduction failure and axonal degeneration in guillain-barré syndrome

To investigate the pathophysiological role of anti-GM1 antibody in Gullain-Barre syndrome (GBS), we reviewed sequential nerve conduction studies of 345 nerves in 34 GBS patients. Statistically significant correlation between IgG anti-GM1 antibodies and electrodiagnoses was found. Sixteen IgG anti-GM1-positive patients were classified as having acute motor sensory axonal neuropathy (AMAN or AMSAN) (12 patients), as having acute inflammatory demyelinating polyneuropathy (AIDP) (3 patientsrpar;, or as undetermined (1 patient) by electrodiagnostic criteria. Besides axonal features, there was rapid resolution of conduction slowing and block. In 3 patients initially diagnosed as having AIDP, conduction slowing was resolved within days, and 1 of them and 3 AMAN patients showed markedly rapid increases in amplitudes of distal compound muscle action potentials that were not accompanied by prolonged duration and polyphasia. The time courses of conduction abnormalities were distinct from those in IgG anti-GM1-negative AIDP patients. Rapid resolution of conduction slowing and block, and the absence of remyelinating slow components, suggest that conduction failure may be caused by impaired physiological conduction at the nodes of Ranvier. Reversible conduction failure as well as axonal degeneration constitutes the pathopsiological mechanisms in IgG anti-GM1)positive GBS. In both cases, immune-mediated attack probably occurs on the axolemma of motor fibers.     

Reduced-intensity unrelated cord blood transplantation for patients with advanced malignant lymphoma.

We report the results of reduced-intensity unrelated cord blood transplantation (RI-UCBT) in patients with advanced malignant lymphoma. Twenty patients (median age, 46.5 years; range, 27-66 years) underwent RI-UCBT with a preparative regimen consisting of fludarabine 125 mg/m2 , melphalan 80 mg/m 2 , and 4 Gy of total body irradiation. The median infused total cell dose was 2.75 x 10(7)/kg (range, 2.3-3.4 x 10(7)/kg). Graft-versus-host disease (GVHD) prophylaxis was composed of cyclosporine or tacrolimus alone. Fifteen patients achieved primary neutrophil engraftment after a median of 20 days. Eight patients developed grade II to IV acute GVHD, and 2 developed chronic GVHD. Of the 16 patients with evaluable disease, 10 achieved a complete response. Primary disease recurred in 1 patient, and transplant-related mortality within 100 days occurred in 8 of 20 patients. The estimated 1-year probability of progression-free survival was 50%. These data suggest that RI-UCBT is a feasible option for patients with refractory lymphoma who lack an HLA-matched donor.     

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.     

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.     

De novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)] with intact blood group-MN locus,confining its locus to 4q28.2–4q31.1

We report a malformed female infant withde novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)]. The MN blood type analysis of the family members showed that the patient had an intact blood group-MN locus. The locus of the gene responsible for the MN antigen activity is confined to a 4q28.2–4q31.1 segment on the basis of the result of this patient and the previous mapping data.