Cloning of a Gene for an Acyl-CoA Dehydrogenase from Pisum sativum L. and Purification and Characterization of Its Product as an Isovaleryl-CoA Dehydrogenase

Isovaleryl-CoA dehydrogenase (IVD, EC 1.3.99.10) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes

Glycosaminoglycans and Sialylated Glycans Sequentially Facilitate Merkel Cell Polyomavirus Infectious Entry

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus

Smad4-expression is decreased in breast cancer tissues: a retrospective study

BACKGROUND: Although transforming growth factor β (TGF-β) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-β signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer. METHODS: Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998. The prognostic value of Smad4-expression was analyzed. RESULTS: Smad4 expression was found to be reduced in lobular and ductal breast carcinoma as compared to surrounding uninvolved lobular and ductal breast epithelia (p < 0.001, n = 50). Smad4-expression correlated positively with expression of TGF-β-receptor I (p < 0.001, n = 197) and TGF-β-receptor II (p < 0.001, n = 197), but showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression. While not achieving statistical significance, there was a trend towards longer survival times in patients with Smad4 negative tumors. CONCLUSION: According to the suggested role of Smad4 as a tumor suppressor we observed that expression of Smad4 is lower in human breast cancer than in surrounding breast epithelium. However, we also observed a trend towards longer survival times in Smad4-negative patients, indicating the complex role of TGF-β signaling in tumor progression

Identifying the Clinical Needs and Patterns of Health Service Use of Adolescent Girls and Women with Autism Spectrum Disorder

Girls and women in the general population present with a distinct profile of clinical needs and use more associated health services compared to boys and men; however, research focused on health service use patterns among girls and women with Autism Spectrum Disorder (ASD) is limited. In the current study, caregivers of 61 adolescent girls and women with ASD and 223 boys and men with ASD completed an online survey. Descriptive analyses were conducted to better understand the clinical needs and associated service use patterns of girls and women with ASD. Sex/gender comparisons were made of individuals’ clinical needs and service use. Adolescent girls and women with ASD had prevalent co-occurring mental and physical conditions and parents reported elevated levels of caregiver strain. Multiple service use was common across age groups, particularly among adolescent girls and women with intellectual disability. Overall, few sex/gender differences emerged, although a significantly greater proportion of girls and women accessed psychiatry and emergency department services as compared to boys and men. Though the current study is limited by its use of parent report and small sample size, it suggests that girls and women with ASD may share many of the same high clinical needs and patterns of services use as boys and men with ASD. Areas for future research are discussed to help ensure appropriate support is provided to this understudied population.Grant sponsor: Canadian Institutes of Health Research; Grant number: MOP 102677; Grant sponsor: Canadian Institutes of Health Research in partnership with NeuroDevNet, Sinneave Family Foundation, CASDA, Autism Speaks Canada, and Health Canada; Grant number: RN162466-28420

Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health

This paper is not subject to U.S. copyright. The definitive version was published in Harmful Algae 14 (2012): 271-300, doi:10.1016/j.hal.2011.10.025.Over the last decade, our understanding of the environmental controls on Pseudo-nitzschia blooms and domoic acid (DA) production has matured. Pseudo-nitzschia have been found along most of the world's coastlines, while the impacts of its toxin, DA, are most persistent and detrimental in upwelling systems. However, Pseudo-nitzschia and DA have recently been detected in the open ocean's high-nitrate, low-chlorophyll regions, in addition to fjords, gulfs and bays, showing their presence in diverse environments. The toxin has been measured in zooplankton, shellfish, crustaceans, echinoderms, worms, marine mammals and birds, as well as in sediments, demonstrating its stable transfer through the marine food web and abiotically to the benthos. The linkage of DA production to nitrogenous nutrient physiology, trace metal acquisition, and even salinity, suggests that the control of toxin production is complex and likely influenced by a suite of environmental factors that may be unique to a particular region. Advances in our knowledge of Pseudo-nitzschia sexual reproduction, also in field populations, illustrate its importance in bloom dynamics and toxicity. The combination of careful taxonomy and powerful new molecular methods now allow for the complete characterization of Pseudo-nitzschia populations and how they respond to environmental changes. Here we summarize research that represents our increased knowledge over the last decade of Pseudo-nitzschia and its production of DA, including changes in worldwide range, phylogeny, physiology, ecology, monitoring and public health impacts

The mucosal immune system and its regulation by autophagy

The gastrointestinal tract presents a unique challenge to the mucosal immune system, which has to constantly monitor the vast surface for the presence of pathogens, while at the same time maintaining tolerance to beneficial or innocuous antigens. In the intestinal mucosa, specialized innate and adaptive immune components participate in directing appropriate immune responses toward these diverse challenges. Recent studies provide compelling evidence that the process of autophagy influences several aspects of mucosal immune responses. Initially described as a “self-eating” survival pathway that enables nutrient recycling during starvation, autophagy has now been connected to multiple cellular responses, including several aspects of immunity. Initial links between autophagy and host immunity came from the observations that autophagy can target intracellular bacteria for degradation. However, subsequent studies indicated that autophagy plays a much broader role in immune responses, as it can impact antigen processing, thymic selection, lymphocyte homeostasis, and the regulation of immunoglobulin and cytokine secretion. In this review, we provide a comprehensive overview of mucosal immune cells and discuss how autophagy influences many aspects of their physiology and function. We focus on cell type-specific roles of autophagy in the gut, with a particular emphasis on the effects of autophagy on the intestinal T cell compartment. We also provide a perspective on how manipulation of autophagy may potentially be used to treat mucosal inflammatory disorders

GABAA receptor subtype involvement in addictive behaviour

GABAA receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarise the evidence that variations in genes encoding GABAA receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABAA receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarise the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABAA Receptor Subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour