Steroidogenic electron transport in adrenal cortex mitochondria

Summary The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxinadrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450_scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450_scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450_scc to which this phospho-lipid binds.     

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry

Objective

Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.

Methods

The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.

Results

Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABA_A) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

Conclusions

Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Avoidance and aggregation create consistent egg distribution patterns of congeneric caddisflies across spatially variable oviposition landscapes

Oecologia - Amongst oviparous animals, the spatial distribution of individuals is often set initially by where females lay eggs, with potential implications for populations and species coexistence....     

ORIGIN AND DIVERSIFICATION OF THE CALIFORNIA FLORA: RE‐EXAMINING CLASSIC HYPOTHESES WITH MOLECULAR PHYLOGENIES

The California Floristic Province exhibits one of the richest floras on the planet, with more than 5500 native plant species, approximately 40% of which are endemic. Despite its impressive diversity and the attention it has garnered from ecologists and evolutionary biologists, historical causes of species richness and endemism in California remain poorly understood. Using a phylogenetic analysis of 16 angiosperm clades, each containing California natives in addition to species found only outside California, we show that CA's current biodiversity primarily results from low extinction rates, as opposed to elevated speciation or immigration rates. Speciation rates in California were lowest among Arcto‐Tertiary lineages (i.e., those colonizing California from the north, during the Tertiary), but extinction rates were universally low across California native plants of all historical, geographic origins. In contrast to long‐accepted ideas, we find that California diversification rates were generally unaffected by the onset of the Mediterranean climate. However, the Mediterranean climate coincided with immigration of many desert species, validating one previous hypothesis regarding origins of CA's plant diversity. This study implicates topographic complexity and climatic buffering as key, long‐standing features of CA's landscape favoring plant species persistence and diversification, and highlights California as an important refuge under changing climates.     

Tuberculosis Case Fatality and Other Causes of Death among Multidrug-Resistant Tuberculosis Patients in a High HIV Prevalence Setting, 2000-2008, South Africa

Introduction

South Africa has the highest reported rates of multi-drug resistant TB in Africa, typified by poor treatment outcomes, attributable mainly to high default and death rates. Concomitant HIV has become the strongest predictor of death among MDR-TB patients, while anti-retroviral therapy (ART) has dramatically reduced mortality. TB Case fatality rate (CFR) is an indicator that specifically reports on deaths due to TB.

Aim

The aim of this paper was to investigate causes of death amongst MDR-TB patients, the contribution of conditions other than TB to deaths, and to determine if causes differ between HIV-uninfected patients, HIV-infected patients receiving ART and those without ART.

Methods

We carried out a retrospective review of data captured from the register of the MDR-TB programme of the North West Province, South Africa. We included 671 patients treated between 2000–2008; 59% of the cohort was HIV-infected and 33% had received ART during MDR treatment. The register contained data on treatment outcomes and causes of death.

Results

Treatment outcomes between HIV-uninfected cases, HIV-infected cases receiving ART and HIV-infected without ART differed significantly (p<0.000). The cohort death rate was 24%, 13% for HIV-uninfected cases and 31% for HIV-infected cases. TB caused most of the deaths, resulting in a cohort CFR of 15%, 9% for HIV-uninfected cases and 20% for HIV-infected cases. Cohort mortality rate due to other conditions was 2%. AIDS-conditions rather than TB caused significantly more deaths among HIV-infected cases receiving ART than those not (p = 0.02).

Conclusions

The deaths among HIV-infected individuals contribute substantially to the high death rate. ART co-therapy protected HIV-infected cases from death due to TB and AIDS-conditions. Mechanisms need to be in place to ensure that HIV-infected individuals are retained in care upon completion of their MDR-TB treatment.     

MegaPlex PCR: a strategy for multiplex amplification

'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by co-amplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.     

Towards an interactive,process‐based approach to understanding range shifts: developmental and environmental dependencies matter

Many species are undergoing distributional shifts in response to climate change. However, wide variability in range shifting rates has been observed across taxa, and even among closely‐related species. Attempts to link climate‐mediated range shifts to traits has often produced weak or conflicting results. Here we investigate interactive effects of developmental processes and environmental stress on the expression of traits relevant to range shifts. We use an individual‐based modelling approach to assess how different developmental strategies affect range shift rates under a range of environmental conditions. We find that under stressful conditions, such as at the margins of the species’ fundamental niche, investment in prolonged development leads to the greatest rates of range shifting, especially when longer time in development leads to improved fecundity and dispersal‐related traits. However, under benign conditions, and when traits are less developmentally plastic, shorter development times are preferred for rapid range shifts, because higher generational frequency increases the number of individual dispersal events occurring over time. Our results suggest that the ability of a species to range shift depends not only on their dispersal and colonisation characteristics but also how these characteristics interact with developmental strategies. Benefits of any trait always depended on the environmental and developmental sensitivity of life history trait combinations, and the environmental conditions under which the range shift takes place. Without considering environmental and developmental sources of variation in the expression of traits relevant to range shifts, there is little hope of developing a general understanding of intrinsic drivers of range shift potential.