Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.
In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2β genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2β genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2β genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.
Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-β signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130757F mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130757F;µMT−/− compound mutant mice, but fibrosis still occurred in their Smad3−/− counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-β/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis.
interleukin 6; pulmonary fibrosis; Smad3; Stat3; transforming growth factor beta
Background and Aims
Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima.
Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization.
The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM.
Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.
Auxin; cytokinins; CDKs; Cucurbita maxima; root apical meristems
This article presents the results of an empirical test of a literature-based Patient-Centered Culturally Sensitive Health Care Model. The model was developed to explain and improve health care for ethnically diverse patients seen in community-based primary care clinics.
Samples of predominantly low-income African American (N = 110) and non-Hispanic White American (N = 119) patients were recruited to complete questionnaires about their perceived health care provider cultural sensitivity and adherence to their provider's treatment regimen recommendations.
Main Outcome Measures
Patients completed written measures of their perceived provider cultural sensitivity, trust in provider, interpersonal control, satisfaction with their health care provider, physical stress and adherence to provider recommended treatment regimen variables (i.e., engagement in a health promoting lifestyle, and dietary and medication adherence).
Two-group path analyses revealed significant links between patient-perceived provider cultural sensitivity and adherence to provider treatment regimen recommendations, with some differences in associations emerging by race/ethnicity.
The findings provide empirical support for the potential usefulness of the Patient-Centered Culturally Sensitive Health Care Model for explaining the linkage between the provision of patient-centered, culturally-sensitive health care, and the health behaviors and outcomes of patients who experience such care.
patient-centered culturally sensitive health care; interpersonal control; health promoting lifestyle; patient satisfaction; treatment adherence
To assess the attitudes of upper-year undergraduate medical students (ie, clerks) toward the philosophy of community inclusion of persons with intellectual disabilities (ID) according to demographic, personal contact, and training variables.
Cross-sectional self-administered survey.
Clerkship rotations at Queen’s University in Kingston, Ont, and the University of Toronto in Ontario in 2006.
A total of 258 clerks.
Main outcome measures
Scores on the Community Living Attitudes Scale—Short Form.
There were no differences in the Community Living Attitudes Scale—Short Form subscale scores across categories of demographic characteristics, personal contact, or having received didactic training about ID. Clerks who had seen patients with ID during their medical school training had higher mean sheltering subscale scores than those who had not (3.27 vs 3.07, P = .02). Additional analysis revealed that 88.5% of clerks who had seen patients with ID reported seeing 5 or fewer such patients, and that those who rated the quality of their supervision more positively had higher mean scores on the empowerment subscale and lower mean scores on the sheltering subscale.
Although specific training has the potential to promote more socially progressive attitudes regarding persons with ID, lower-quality supervision is associated with higher endorsement of items expressing the need to shelter individuals with ID from harm and lower endorsement of items promoting empowerment.
SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.
During the 2010 cholera outbreak in Haiti, water and seafood samples were collected to detect Vibrio cholerae. The outbreak strain of toxigenic V. cholerae O1 serotype Ogawa was isolated from freshwater and seafood samples. The cholera toxin gene was detected in harbor water samples.
Vibrio cholerae; cholera; drinking water; seafood safety; ultrafiltration
Parkinson’s disease [PD], a progressive neurodegenerative disease, results in abnormal accumulation of insoluble alpha-synuclein [α-Syn] in dopaminergic neurons. Here we examined tauopathic changes and the α-Syn/p-GSK-3β/proteasome pathway in postmortem striata and inferior frontal gyri [IFG] from patients with PD and PD with dementia [PDD]. In both PD and PDD, α-Syn levels were high, especially the insoluble form of this protein; in PDD, insoluble α-Syn levels were persistently higher than PD across both brain regions. Levels of p-GSK-3β phosphorylated at Tyr 216, which hyperphosphorylates Tau to produce toxic pathological forms of p-Tau, were higher in striata of both PD and PDD compared to controls, but were unaltered in IFG. While proteasomal activity was unchanged in striatum of PD and PDD, such activity was diminished in the IFG of both PD and PDD. A decrease in 19S subunit of the proteasomes was seen in IFG of PDD, while lower levels of 20S subunits were seen in striatum and IFG of both PD and PDD patients. Parkin levels were similar in PD and PDD, suggesting lack of involvement of this protein. Most interestingly, tauopathic changes were noted only in striatum of PD and PDD, with increased hyperphosphorylation seen at Ser262 and Ser396/404; increases in Ser202 levels were seen only in PD but not in PDD striatum. We were unable to detect any tauopathy in IFG in either PD or PDD despite increased levels of α-Syn, and decreased proteasomal activity, and is probably due to lack of increase in p-GSK-3β in IFG. Unlike Alzheimer’s disease where tauopathy is more globally observed in diverse brain regions, our data demonstrates restricted expression of tauopathy in brains of PD and PDD, probably limited to dopaminergic neurons of the nigrostriatal region.
synucleinopathies; tauopathies; neurodegeneration; Alzheimer’s disease; dementia; hyperphosphorylated Tau; p-GSK-3β; parkin; neurofibrillary tangles; Lewy bodies
V(D)J recombination, the process that rearranges gene segments to assemble mature antigen receptor genes, relies on a recombinase comprising the RAG1 and RAG2 proteins. RAG1 is a multi-functional enzyme including DNA binding and cleavage as well as ubiquitin ligase activities, all of which appear to contribute to its role in recombination. Here we demonstrate that components of the ubiquitin conjugation machinery and the 26S proteasome are required for an early step in V(D)J recombination. Inhibitors of the 26S proteasome and ubiquitin activating enzyme (E1) blocked both chromosomal and extra-chromosomal recombination when added one hour following transfection/induction, but they had no effect when added 16 hours later. There was no effect on expression of RAG1, and recombination did not require transit through the cell cycle, confirming that inhibition was not due to an indirect effect on cell cycle arrest or protein expression. Experiments in which RAG1 translation was blocked with cyclohexamide after 16 hours of expression indicated that many active recombination complexes were formed within this window, although recombination products continued to accumulate for 48 hours. These data suggest that ubiquitin-dependent degradation is an early step in complex assembly or activation, and are consistent with our previous hypothesis that degradation of a negative regulator is required to trigger recombination.
V(D)J recombination; RAG1; 26S proteasome; ubiquitin activating enzyme; E1
Fatigue is a symptom of acute myocardial infarction (AMI); however, few studies have characterized the fatigue associated with AMI in men and women.
The convenience sample included 88 men and 28 women admitted with a diagnosis of AMI at 6 Midwestern facilities. Data were collected upon hospital admission and 30 days after discharge. A total of 37 men and 10 women responded to the 30-day questionnaires. The Profile of Mood States Fatigue (POMS-F) subscale was used to measure fatigue and the Depression-Dejection subscale to measure depressed mood.
At baseline, significant gender differences were found, with women more likely to have higher POMS-F scores (15.80, SD = 7.33) than men (11.19, SD = 7.04, P = .004). Significant gender differences were also found at 30 days (t = 2.40, df = 45, P = .02). POMS-F scores for women were higher at baseline, with decreased fatigue levels 30 days after discharge (t = 5.36, df = 9, P ≤ .05). No differences were found in POMS-F scores for men (t = 1.26, df = 36, P = .213) between baseline and 30 days after discharge. Fatigue was associated with depressed mood and gender at baseline (R2 = 0.48, P < .05) and 30 days after discharge (R2 = 0.308, P < .05).
In this sample, fatigue at baseline and at 30 days after discharge was associated with gender and depressed mood. Women reported high levels of fatigue with AMI and lower fatigue after discharge. Men reported moderate to high fatigue levels, which did not change over time. Further research is needed to discern fatigue patterns before and after AMI.
acute myocardial infarction; fatigue; gender differences; symptoms; women
The purpose of this study was to evaluate the psychometric properties of three questionnaires to measure fatigue with myocardial infarction. The Fatigue Symptom Inventory Interference Scale, Profile of Moods States Fatigue subscale (POMS-F), and Short Form 36 (SF-36) Vitality Scale were completed during hospitalization (n = 116) and 30 days after hospital admission (n = 49). Moderate to strong correlations were found among each of these fatigue scales and between each fatigue scale and measures of other variables to include vigor, depressed mood, anxiety, and physical functioning. POMS-F scores decreased significantly at Time 2, but this decline in fatigue was not validated on the other fatigue scales. Patients’ Time 1 scores reflected significantly more fatigue compared to published scores for healthy adults. The ability to discriminate between groups suggests that the instruments may be useful for identifying patients with cardiovascular risk factors who report clinically significant fatigue.
cardiovascular; fatigue; descriptive quantitative; acute care
Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.
The RAG1 recombinase, which participates in DNA manipulation during rearrangement of antigen receptor genes in developing immune cells, possesses ubiquitin ligase activity. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1. While RAG1 auto-ubiquitylation is dependent on the ubiquitin conjugating enzyme CDC34, ubiquitylation of KPNA1 was best supported by UbcH2/Rad6 and UbcH5a. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. This is the first putative substrate identified for the RAG1 ubiquitin ligase, and to our knowledge it is the first reported case of ubiquitylation of KPNA1.
We present results of a feasibility test of a sequential treatment strategy using continuation phase cognitive-behavioral therapy (CBT) to prevent relapse in youths with major depressive disorder (MDD) who have responded to acute phase pharmacotherapy.
Forty-six youths (ages 11–18 years) who had responded to 12 weeks of treatment with fluoxetine were randomized to receive either 6 months of continued antidepressant medication management (MM) or antidepressant MM plus relapse prevention CBT (MM+CBT). Primary outcome was time to relapse, defined as a Childhood Depression Rating Scale-Revised score of 40 or higher and 2 weeks of symptom worsening or clinical deterioration warranting alteration of treatment to prevent full relapse.
Cox proportional hazards regression, adjusting for depression severity at randomization and for the hazard of relapsing by age across the trial, revealed that participants in the MM treatment group had a significantly greater risk for relapse than those in the MM+CBT treatment group (hazard ratio = 8.80; 95% confidence interval 1.01–76.89; χ2 = 3.86, p = .049) during 6 months of continuation treatment. In addition, patient satisfaction was significantly higher in the MM+CBT group. No differences were found between the two treatment groups on attrition rate, serious adverse events, and overall global functioning.
These preliminary results suggest that continuation phase CBT reduces the risk for relapse by eightfold compared with pharmacotherapy responders who received antidepressant medication alone during the 6-month continuation phase.
depression; CBT; relapse prevention; sequential treatment
The third meeting of the Cognitive Neuroscience Treatment Research to Improve Cognition in Schizophrenia (CNTRICS) was focused on selecting promising measures for each of the cognitive constructs selected in the first CNTRICS meeting. In the domain of working memory, the 2 constructs of interest were goal maintenance and interference control. CNTRICS received 3 task nominations for each of these constructs, and the breakout group for working memory evaluated the degree to which each of these tasks met prespecified criteria. For goal maintenance, the breakout group for working memory recommended the AX-Continuous Performance Task/Dot Pattern Expectancy task for translation for use in clinical trial contexts in schizophrenia research. For interference control, the breakout group recommended the recent probes and operation/symmetry span tasks for translation for use in clinical trials. This article describes the ways in which each of these tasks met the criteria used by the breakout group to recommend tasks for further development.
inference control; goal maintenance; schizophrenia
Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.
BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.
Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.
Smoke induced inflammation does not protect against influenza infection.
In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.
OBJECTIVE: To develop and test the reliability of three race/ethnicity-specific forms of the pilot Tucker-Culturally Sensitive Health Care Inventory (T-CUSHCI) for use by patients at community-based primary care centers to evaluate the level of patient-centered cultural sensitivity perceived in the health care that they experience. METHODS: This research involved two studies using independent samples of primary care patients. In study 1, mostly low-income African-American, Hispanic and non-Hispanic white American patients (N=221) rated the importance of specific provider and office staff behaviors and attitudes, and center policies and physical environment characteristics that were earlier identified in previous focus groups as characteristics of patient-centered culturally sensitive healthcare. In study 2, three pilot race/ethnicity-specific T-CUSHCI patient forms were constructed from the items rated as at least important in study 1. Mostly low-income African-American and non-Hispanic white American patients (N=180) provided data to determine the reliability of the T-CUSHCI patient form for their racial/ethnic group. RESULTS: The pilot T-CUSHCI-African-American patient form and the pilot T-CUSHCI-non-Hispanic white American patient form were found to have Cronbach's alpha coefficients ranging from 0.71-0.96 and six-month test-retest and split-half reliabilities ranging from 0.92-0.99. CONCLUSION: The pilot T-CUSHCI patient forms (one each for African Americans, Hispanics and non-Hispanic whites) should be further tested using a national sample of patients. In the interim, these inventory forms can be used as clinical tools to obtain patient feedback for providing "individualized" patient-centered culturally sensitive healthcare.
Recent findings have established an association between obesity and immune dysfunction. However, most of the studies investigating the effects of obesity on immune function have been carried out in genetically obese rodent models. Since human obesity is mostly due to intake of a high fat diet and decreased energy expenditure, we asked whether immunological defects also occur in diet-induced obesity. Specifically, we focused on the function of monocytes and macrophages, as these cells are thought to be involved in the low-grade inflammation present in obesity.
Male Sprague-Dawley rats were fed a high-fat or a standard chow diet for either 2 or 10 weeks. At the end of the intervention period animals were anaesthetised, blood collected for determination of plasma mediator concentrations and lipopolysaccharide (LPS) stimulated production of TNF-α by monocytes. LPS stimulated production of TNF-α in alveolar macrophages was also determined.
High-fat feeding for either 2 or 10 weeks resulted in significant increases in fat mass and serum leptin. Although increased serum leptin has previously been linked to modulation of innate immunity, we found no significant difference in the LPS stimulated production of TNF-α by either blood monocytes or alveolar macrophages between the dietary groups. Furthermore, we failed to find a significant increase in circulating TNF-α concentrations in obese animals, as reported for genetically obese animals.
Our data suggest that defects in innate immune function observed in genetically obese animals are not mimicked by dietary obesity, and may more likely reflect the gross abnormality in leptin function of these models. Further work is required delineate the effects of dietary obesity on inflammatory state and immune function.
innate immunity; leptin; lipopolysaccharide; macrophage; neuropeptide Y; obesity; tumour necrosis factor