Docking of endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane: a pentabasic amino acid sequence in the autoinhibitory domain of eNOS targets a proteinase K-cleavable peptide on the cytoplasmic face of mitochondria

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628-632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.     

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.     

Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs.     

Time-Course Proteome Analysis Reveals the Dynamic Response of Cryptococcus gattii Cells to Fluconazole

Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC.     

3-methyladenine DNA glycosylases: structure,function, and biological importance

The genome continuously suffers damage due to its reactivity with chemical and physical agents. Finding such damage in genomes (that can be several million to several billion nucleotide base pairs in size) is a seemingly daunting task. 3-Methyladenine DNA glycosylases can initiate the base excision repair (BER) of an extraordinarily wide range of substrate bases. The advantage of such broad substrate recognition is that these enzymes provide resistance to a wide variety of DNA damaging agents; however, under certain circumstances, the eclectic nature of these enzymes can confer some biological disadvantages. Solving the X-ray crystal structures of two 3-methyladenine DNA glycosylases, and creating cells and animals altered for this activity, contributes to our understanding of their enzyme mechanism and how such enzymes influence the biological response of organisms to several different types of DNA damage. BioEssays 21:668–676, 1999. © 1999 John Wiley & Sons, Inc.     

Anticoccidial Activity of Berberine against Eimeria-Infected Chickens

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.     

Changes in health indicator gaps between First Nations and other residents of Manitoba

Background:The Truth and Reconciliation Commission of Canada has called for better reporting of health disparities between First Nations people and other Canadians to close gaps in health outcomes. We sought to evaluate changes in these disparities using indicators of health and health care use over the last 2 decades.Methods:We used linked, whole-population, administrative claims data from the Manitoba Centre for Health Policy for fiscal years 1994/95 to 1998/99 and 2012/13 to 2016/17. We measured indicators of health and health care use among registered First Nations and all other Manitobans, and compared differences between these groups over the 2 time periods.Results:Over time, the relative gap between First Nations and all other Manitobans widened by 51% (95% confidence interval [CI] 42% to 60%) for premature mortality rate. For potential years of life lost, the gap widened by 54% (95% CI 51% to 57%) among women and by 32% (95% CI 30% to 35%) among men. The absolute gap in life expectancy widened by 3.14 years (95% CI 2.92 to 3.36) among men and 3.61 years (95% CI 3.38 to 3.84) among women. Relative gaps widened by 20% (95% CI 12% to 27%) for ambulatory specialist visits, by 14% (95% CI 12% to 16%) for hospital separations and by 50% (95% CI 39% to 62%) for days spent in hospital, but narrowed by 33% (95% CI −36% to −30%) for ambulatory primary care visits, by 22% (95% CI −27% to −16%) for mammography and by 27% (95% CI −40% to −23%) for injury hospitalizations.Interpretation:Disparities between First Nations and all other Manitobans in many key indicators of health and health care use have grown larger over time. New approaches are needed to address these disparities and promote better health with and for First Nations.

Comprehensive data on the health of First Nations people in Canada are urgently needed.^1,2 This is a priority identified by the Truth and Reconciliation Commission of Canada, which highlights the striking health disparities between Indigenous and non-Indigenous populations in Canada and recognizes the disparities in their Calls to Action as “a direct result of previous Canadian government policies.”³ These health disparities are understood to be part of the continuing impact of colonization and genocidal policies aimed specifically at Indigenous people.^4,5The underlying causes of the health gap between Indigenous and non-Indigenous people, resulting from attempts by European settlers to assimilate Indigenous people into their own societies, have been detailed elsewhere.⁶ The collective trauma that Indigenous populations have experienced through colonial policies and practices include, among others, racism and marginalization in virtually every aspect of their lives; major disruptions of families and communities through forced attendance at residential schools and by the child welfare system; trauma from physical, emotional and sexual abuse, carried over into future generations; and damage to their Indigenous identity through loss of culture, language, traditions and teachings.^6,7 Other social determinants (e.g., poverty, social exclusion and poor access to clean water, quality housing, education and heath services) and the governments’ failure to address these issues also influence the health of First Nations people.^8,9A previous population-based study of First Nations’ health and health care use in Canada, published in 2002, focused on the Manitoba population, which has the highest proportion of First Nations residents among Canadian provinces.¹⁰ It identified substantial gaps in the health of First Nations and their access to health services compared with other Manitobans. Others have since reported on health disparities between Indigenous peoples and other Canadians;^11–14 however, the Manitoba data provide a unique opportunity to compare changes in health-related outcomes over nearly 2 decades. In the present study, we sought to compare indicators of health and health care use between First Nations people and other residents of Manitoba, to determine whether the gap between the 2 groups has changed over the past 18 years and establish whether any progress has been made in improving First Nations’ health.     

Pulsed Estrogen Therapy Prevents Post-OVX Porcine Dura Mater Microvascular Network Weakening via a PDGF-BB-Dependent Mechanism

In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurysm, and decline in cognitive abilities. In younger postpartum women and those using oral contraceptives, perturbations in E2 are associated with higher risk of cerebral venous thrombosis. A number of serious intracranial pathologic conditions linked to E2 deficiencies, such as dural sinus thrombosis, dural fistulae, non-parenchymal intracranial hemorrhages, migraines, and spontaneous cerebrospinal fluid leaks, involve the vessels not of the brain itself, but of the outer fibrous membrane of the brain, the dura mater (DM). The pathogenesis of these disorders remains mysterious and how estrogen regulates structural and functional integrity of DM vasculature is largely unknown. Here, we demonstrate that post ovariectomy (OVX) DM vascular remodeling is manifested by microvessel destabilization, capillary rarefaction, increased vascular permeability, and aberrant angio-architecture, and is the result of disrupted E2-regulated PDGF-BB signaling within dura microvasculature. These changes, associated with the reduction in systemic PDGF-BB levels, are not corrected by a flat-dose E2 hormone replacement therapy (HRT), but are largely prevented using HRT schedules mimicking physiological E2 fluctuations. We demonstrate that 1) E2 regulates PDGF-BB production by endothelial cells in a dose-dependent manner and 2) optimization of PDGF-BB levels and induction of robust PDGF-mediated endothelial cell-vascular pericyte interactions require high (estrous) E2 concentrations. We conclude that high (estrous) levels of E2 are important in controlling PDGF-mediated crosstalk between endothelial cells and pericytes, a fundamental mechanism governing microvessel stability and essential for preserving intracranial homeostasis.