Effects of ANK3 variation on gray and white matter in bipolar disorder.

The single-nucleotide polymorphism rs9804190 in the Ankyrin G (ANK3) gene has been reported in genome-wide association studies to be associated with bipolar disorder (BD). However, the neural system effects of rs9804190 in BD are not known. We investigated associations between rs9804190 and gray and white matter (GM and WM, respectively) structure within a frontotemporal neural system implicated in BD. A total of 187 adolescent and adult European Americans were studied: a group homozygous for the C allele (52 individuals with BD and 56 controls) and a T-carrier group, carrying the high-risk T allele (38 BD and 41 controls). Subjects participated in high-resolution structural magnetic resonance imaging and diffusion tensor imaging (DTI) scanning. Frontotemporal region of interest (ROI) and whole-brain exploratory analyses were conducted. DTI ROI-based analysis revealed a significant diagnosis by genotype interaction within the uncinate fasciculus (P⩽0.05), with BD subjects carrying the T (risk) allele showing decreased fractional anisotropy compared with other subgroups, independent of age. Genotype effects were not observed in frontotemporal GM volume. These findings support effects of rs9804190 on frontotemporal WM in adolescents and adults with BD and suggest a mechanism contributing to WM pathology in BD.

Genetic epilepsy with febrile seizures plus: definite and borderline phenotypes.

Generalised epilepsy with febrile seizures plus (GEFS+) is the most studied familial epilepsy syndrome. However, characteristics of UK families have not previously been reported. Among the first 80 families recruited to our families study, four broad subphenotypes were identified: families with classical GEFS+; families with borderline GEFS+; families with unclassified epilepsy; and families with an alternative syndromal diagnosis. Borderline GEFS+ families shared many characteristics of classical GEFS+ families-such as prominent febrile seizures plus and early onset febrile seizures-but included more adults with focal epilepsies (rather than the idiopathic generalised epilepsies predominating in GEFS+) and double the prevalence of migraine. Thus the authors believe that a novel and robust familial epilepsy phenotype has been identified. Subcategorising families with epilepsy is helpful in targeting both clinical and research resources. Most families with GEFS+ have no identified causal mutation, and so predicting genetic homogeneity by identifying endophenotypes becomes more important.

The role of Cathepsin S as a marker of prognosis and predictor of chemotherapy benefit in adjuvant CRC: a pilot study.

BACKGROUND: The aim of this pilot retrospective study was to investigate the immunohistochemical expression of Cathepsin S (CatS) in three cohorts of colorectal cancer (CRC) patients (n=560). METHODS: Prevalence and association with histopathological variables were assessed across all cohorts. Association with clinical outcomes was investigated in the Northern Ireland Adjuvant Chemotherapy Trial cohort (n=211), where stage II/III CRC patients were randomised between surgery-alone or surgery with adjuvant fluorouracil/folinic acid (FU/FA) treatment. RESULTS: Greater than 95% of tumours had detectable CatS expression with significantly increased staining in tumours compared with matched normal colon (P>0.001). Increasing CatS was associated with reduced recurrence-free survival (RFS; P=0.03) among patients treated with surgery alone. Adjuvant FU/FA significantly improved RFS (hazard ratio (HR), 0.33; 95% CI, 0.12-0.89) and overall survival (OS; HR, 0.25; 95% CI, 0.08-0.81) among 36 patients with high CatS. Treatment did not benefit the 66 patients with low CatS, with a RFS HR of 1.34 (95% CI, 0.60-3.19) and OS HR of 1.33 (95% CI, 0.56-3.15). Interaction between CatS and treatment status was significant for RFS (P=0.02) and OS (P=0.04) in a multivariate model adjusted for known prognostic markers. CONCLUSION: These results signify that CatS may be an important prognostic biomarker and predictive of response to adjuvant FU/FA in CRC.

Adherence and persistence with teriparatide among patients with commercial, Medicare, and Medicaid insurance.

UNLABELLED: Adherence to, and persistence with, treatments for osteoporosis are low. Adherence with teriparatide decreases over time. Higher copayments in the commercial/Medicare population were associated with worse persistence. Understanding factors such as prior screening, prior treatment history, and out of pocket costs that influence persistence with teriparatide may help clinicians make informed decisions. INTRODUCTION: The purpose of this study was to evaluate adherence and persistence with teriparatide. METHODS: Beneficiaries with at least one claim for teriparatide in 2003 or 2004 and continuous enrollment in the previous 12 months and subsequent 6 months were identified in a national commercial/Medicare and Medicaid administrative claims database (MarketScan®). Adherence was assessed through calculation of the medication possession ratio (MPR). Persistence was measured by time until discontinuation and time until first 60-day gap in treatment. Factors associated with persistence were assessed using Cox proportional hazards models. RESULTS: The average MPR at 6 months was 0.74 (N=2,218) and at 12 months, was 0.66 (N=1,303). At 6 months, 64.6% of patients remained on therapy and at 12 months, 56.7% remained. Bone mineral density screening and use of antiresorptive therapy within the 12 months pre-period, and lower patient copayments were associated with increased persistence. CONCLUSION: Patients appear to have good adherence with teriparatide over the first 6 months which declines over time. Prior screening and treatment of osteoporosis and out of pocket costs appear to impact persistence. To optimize patient outcomes, clinicians should consider clinical factors that impact persistence, while healthcare decision makers should consider the negative effect of higher patient copayments on persistence.

Tyrosine-phosphorylated SOCS-3 inhibits STAT activation but binds to p120 RasGAP and activates Ras.

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.

Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7.

The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL-4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor.

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12.

Interleukin (IL-12) has many effects on the function of natural killer and T cells, and is important in the control of cell-mediated immunity. IL-2 and IL-12 display many similar activities, yet each also induces a distinct set of responses. A human IL-12 receptor subunit has recently been cloned and, like the IL-2R beta and IL-2R gamma, is a member of the hematopoietic receptor superfamily; however, the molecular mechanisms of IL-12 action are unknown. In this report we show that IL-12 and IL-2 induce tyrosine phosphorylation of distinct members of the Janus (JAK) family of protein tyrosine kinases in human T lymphocytes. IL-12, but not IL-2, stimulates the tyrosine phosphorylation of TYK2 and JAK2, whereas JAK1 and JAK3, which are phosphorylated in response to IL-2, are not phosphorylated after IL-12 treatment. The use of distinct but related JAK family tyrosine kinases by IL-12 and IL-2 may provide a biochemical basis for their different biological activities.

In vitro correction of JAK3-deficient severe combined immunodeficiency by retroviral-mediated gene transduction.

Mutations affecting the expression of the Janus family kinase JAK3 were recently shown to be responsible for autosomal recessive severe combined immunodeficiency (SCID). JAK3-deficient patients present with a clinical phenotype virtually indistinguishable from boys affected by X-linked SCID, a disease caused by genetic defects of the common gamma chain (gamma c) that is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. The specific interaction of JAK3 and gamma c represents the biochemical basis for the similarities between these two immunodeficiencies. Both forms of SCID are characterized by recurrent, severe infections leading to death in infancy unless successfully treated by allogeneic bone marrow transplantation. Because of the potentially lethal complications associated with allogeneic bone marrow transplantation and the frequent lack of suitable marrow donors, the development of alternative forms of therapy is highly desirable. To this end, we investigated a retroviral-mediated gene correction approach for JAK3-deficiency. A vector carrying a copy of JAK3 cDNA was constructed and used to transduce B cell lines derived from patients with JAK3-deficient SCID. We demonstrate restoration of JAK3 expression and phosphorylation upon IL-2 and IL-4 stimulation. Furthermore, patients' cells transduced with JAK3 acquired the ability to proliferate normally in response to IL-2. These data indicate that the biological defects of JAK3-deficient cells can be efficiently corrected in vitro by retroviral-mediated gene transfer, thus providing the basis for future investigation of gene therapy as treatment for JAK3-deficient SCID.

Awake seizures after pure sleep-related epilepsy: a systematic review and implications for driving law.

Who with sleep seizures is safe to drive? Driving law is controversial; ineligibility varies between individual US states and EU countries. Current UK driving law is strongly influenced by a single-centre study from 1974 where most participants were not taking antiepileptic drugs (AEDs). However, pure sleep-related epilepsy is often fully controlled on medication, and its withdrawal can provoke awake seizures. This systematic review asked, 'What is the risk of awake seizures in pure sleep-related epilepsy?' 9885 titles were identified; 2312 were excluded (not human or adult); 40 full texts were reviewed; six papers met our inclusion criteria; each of these six studies had a different pure sleep-related epilepsy definition. Using the largest prospective study, we were able to calculate next year's awake seizure chance (treated with antiepileptic medication). This was maximal in the second year: 5.7% (95% CI 3.0 to 10.4%). European licensing bodies including the UK's Driver and Vehicle Licensing Agency broadly accept a risk of less than 20% for Group 1 licensing. However, this study excluded patients with frontal-lobe epilepsies. Furthermore, follow-up (n=160) varied from 2 to 6 years, yet new awake seizures may occur even after 10-20 years of pure sleep-related epilepsy A paucity of evidence underpins present licensing law; current rulings would be difficult to defend if legally challenged. The law may be penalising people with pure sleep-related epilepsy without increased risk of awake seizures, while failing to identify subgroups at unacceptable risk of an awake seizure at the wheel.

Monitoring fusarium crown rot populations in spring wheat residues using quantitative real-time polymerase chain reaction.

Caused by a complex of Fusarium species including F. culmorum, F. graminearum, and F. pseudograminearum, Fusarium crown rot (FCR) is an important cereal disease worldwide. For this study, Fusarium population dynamics were examined in spring wheat residues sampled from dryland field locations near Bozeman and Huntley, MT, using a quantitative real-time polymerase chain reaction (qPCR) Taqman assay that detects F. culmorum, F. graminearum, and F. pseudograminearum. Between August 2005 and June 2007, Fusarium populations and residue decomposition were measured eight times for standing stubble (0 to 20 cm above the soil surface), lower stem (20 to 38 cm), middle stem (38 to 66 cm), and chaff residues. Large Fusarium populations were found in stubble collected in August 2005 from F. pseudograminearum-inoculated plots. These populations declined rapidly over the next 8 months. Remnant Fusarium populations in inoculated stubble were stable relative to residue biomass from April 2006 until June 2007. These two phases of population dynamics were observed at both locations. Relative to inoculated stubble populations, Fusarium populations in other residue fractions and from noninoculated plots were small. In no case were FCR species observed aggressively colonizing noninfested residues based on qPCR data. These results suggest that Fusarium populations are unstable in the first few months after harvest and do not expand into noninfested wheat residues. Fusarium populations remaining after 8 months were stable for at least another 14 months in standing stubble providing significant inoculums for newly sown crops.

A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase.

A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.

Aggresomes: a cellular response to misfolded proteins.

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.

Optical differentiation of amoebic ectoplasm and endoplasmic flow.

Optical activity differentiating the flowing and nionflowing amoebic cytoplasm was detected. This evidence indicates mtiolecular alignment in the flow stream and can be used to provide data on the direction of alignmtient. The results were obtained by utilizing a dynamnic polarized-light detection system which is sensitive only to specimenis which possess a preferred axis.

Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3: implications for XSCID and XCID.

Interleukin-2 (IL-2) signaling requires the dimerization of the IL-2 receptor beta.(IL-2R beta) and common gamma (gamma c) chains. Mutations of gamma c can result in X-linked severe combined immunodeficiency (XSCID). IL-2, IL-4, IL-7 (whose receptors are known to contain gamma c), and IL-9 (whose receptor is shown here to contain gamma c) induced the tyrosine phosphorylation and activation of the Janus family tyrosine kinases Jak1 and Jak3. Jak1 and Jak3 associated with IL-2R beta and gamma c, respectively; IL-2 induced Jak3-IL-2R beta and increased Jak3-gamma c associations. Truncations of gamma c, and a gamma c, point mutation causing moderate X-linked combined immunodeficiency (XCID), decreased gamma c-Jak3 association. Thus, gamma c mutations in at least some XSCID and XCID patients prevent normal Jak3 activation, suggesting that mutations of Jak3 may result in an XSCID-like phenotype.

Alpha7 nicotinic acetylcholine receptor gene and reduced risk of Alzheimer's disease.

BACKGROUND: Sporadic Alzheimer's disease (AD) is a common disabling disease of complex aetiology for which there are limited therapeutic options. We sought to investigate the role of the alpha7 nicotinic acetylcholine receptor gene (CHRNA7) in influencing risk of AD in a large population. CHRNA7 is a strong candidate gene for AD for several reasons: (1) its expression is altered differentially in the AD brain; (2) it interacts directly with beta amyloid peptide (Abeta(42)); and (3) agonist activation induces several neuroprotective pathways. METHODS: In this study we used a genetic haplotype approach to assess the contribution of common variation at the CHRNA7 locus to risk of AD. Fourteen single nucleotide polymorphisms (SNPs) were genotyped in 764 AD patients and 314 controls. RESULTS: Three blocks of high linkage disequilibrium (LD) and low haplotype diversity were identified. The block 1 TCC haplotype was significantly associated with reduced odds of AD (p = 0.001) and was independent of apolipoprotein E (APOE) status. Individual SNPs were not associated with risk for AD. CONCLUSIONS: We conclude that genetic variation in CHRNA7 influences susceptibility to AD. These results provide support for the development of alpha7nAChR agonists or modulators as potential drug treatments for AD. Further work is necessary to replicate the findings in other populations.

Signaling by type I and II cytokine receptors: ten years after.

Discovered during the past ten years, Janus kinases and signal transducers and activators of transcription have emerged as critical elements in cytokine signaling and immunoregulation. Recently, knockout mice for all the members of these families have been generated, with remarkably specific outcomes. Equally exciting is the discovery of a new class of inhibitors, the suppressor of cytokine signaling family. The phenotypes of mice deficient in these molecules are also striking, underscoring the importance of negative regulation in cytokine signaling.

SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation.

Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop.

Elevated platelet beta-secretase activity in mild cognitive impairment.

BACKGROUND/AIMS: We have recently reported that platelet activity of the rate-limiting enzyme for beta-amyloid peptide production is elevated in established Alzheimer's disease. Laboratory investigation of the very early stages of dementia provides an opportunity to investigate pathological mechanisms before advanced disease hinders interpretation. Mild cognitive impairment (MCI) exists prior to obvious dementia, and is associated with increased risk of conversion to overt disease. METHODS: We developed and used a fluorimetric assay to quantify platelet membrane beta-secretase activity in 52 patients with MCI and 75 controls. RESULTS: Platelet membrane beta-secretase activity was 24% higher in individuals with MCI compared to controls (p = 0.001, unpaired t test with Welch correction). CONCLUSION: Elevated platelet beta-secretase activity in subjects with MCI is an area for further study in relation to the etiology and diagnosis of MCI.

Amyloid precursor protein heat shock response in lymphoblastoid cell lines bearing presenilin-1 mutations.

The amyloid precursor protein (APP) gene promoter contains a heat shock element. An abnormal APP heat shock response could increase accumulation of A beta, the APP metabolite found in Alzheimer's disease amyloid plaques. Since A beta production is affected by presenilin-1 (PS-1) mutations, we investigated whether basal APP levels or response to heat shock were altered in lymphoblastoid cell lines from 8 PS-1 mutation-bearers and 9 control members of Alzheimer's disease families. Lymphoblastoid cell lines were incubated at 42 degrees C for 35 min and allowed to recover at 37 degrees C for 1, 3, 8, 24 and 48 h. APP mRNA levels, quantified using RNA-RNA solution hybridisation, increased significantly at 1 and 3 h post-heat shock to between 123% and 163% of pre-heat shock (0 h) levels and returned to normal by 8 h. Semi-quantitative Western immunoblotting of cell lysates using the 22C11 antibody detected two major bands, migrating at approximately 145 and approximately 120 kDa. Band optical densities increased significantly at 3 h to approximately 155% of 0 h levels, following the increase in APP mRNA levels and showing a similar reversibility. APP mRNA and protein responses were comparable in the PS-1 mutation-bearing and control cell lines. This study shows that both APP mRNA and protein are induced in lymphoblastoid cell lines following heat shock and that this response is not affected by PS-1 mutations which are pathogenic for Alzheimer's disease.