ARL2 and BART Enter Mitochondria and Bind the Adenine Nucleotide Transporter

The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding partner binder of ARL2 (BART) are ubiquitously expressed in rodent and human tissues and are most abundant in brain. Both ARL2 and BART are predominantly cytosolic, but a pool of each was found associated with mitochondria in a protease-resistant form. ARL2 was found to lack covalent N-myristoylation, present on all other members of the ARF family, thereby preserving the N-terminal amphipathic alpha-helix as a potential mitochondrial import sequence. An overlay assay was developed to identify binding partners for the BART.ARL2.GTP complex and revealed a specific interaction with a protein in bovine brain mitochondria. Purification and partial microsequencing identified the protein as an adenine nucleotide transporter (ANT). The overlay assay was performed on mitochondria isolated from five different tissues from either wild-type or transgenic mice deleted for ANT1. Results confirmed that ANT1 is the predominant binding partner for the BART.ARL2.GTP complex and that the structurally homologous ANT2 protein does not bind the complex. Cardiac and skeletal muscle mitochondria from ant1(-)/ant1(-) mice had increased levels of ARL2, relative to that seen in mitochondria from wild-type animals. We conclude that the amount of ARL2 in mitochondria is subject to regulation via an ANT1-sensitive pathway in muscle tissues.     

New Recombination Methods for Sinorhizobium meliloti Genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

Postdischarge thromboprophylaxis and mortality risk after hip-or knee-replacement surgery

Background

Patients undergoing hip or knee replacement are at high risk of developing a postoperative venous thromboembolism even after discharge from hospital. We sought to identify hospital and patient characteristics associated with receiving thromboprophylaxis after discharge and to compare the risk of short-term mortality among those who did or did not receive thromboprophylaxis.

Methods

We conducted a retrospective cohort study using system-wide hospital discharge summary records, physician billing information, medication reimbursement claims and demographic records. We included patients aged 65 years and older who received a hip or knee replace ment and who were discharged home after surgery.

Results

In total we included 10 744 patients. Of these, 7058 patients who received a hip replacement and 3686 who received a knee replacement. The mean age was 75.4 (standard deviation [SD] 6.8) years and 38% of patients were men. In total, 2059 (19%) patients received thomboprophylaxis at discharge. Patients discharged from university teaching hospitals were less likely than those discharged from community hospitals to received thromboprophylaxis after discharge (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.80–1.00). Patients were less likely to receive thromboprophylaxis after discharge if they had a longer hospital stay (15–30 days v. 1–7 days, OR 0.69, 95% CI 0.59–0.81). Patients were more likely to receive thromboprophylaxis if they had hip (v. knee) replacement, osteoarthritis, heart failure, atrial fibrillation or hypertension, higher (v. lower) income or if they were treated at medium-volume hospitals (69–116 hip and knee replacements per year). In total, 223 patients (2%) died in the 3-month period after discharge. The risk of short-term mortality was lower among those who received thromboprophylaxis after discharge (hazard ratio [HR] 0.34, 95% CI 0.20–0.57).

Interpretation

Fewer than 1 in 5 elderly patients discharged home after a hip-or knee-replacement surgery received postdischarge thromboprophylaxis. Those prescribed these medications had a lower risk of short-term mortality. The benefits of and barriers to thromboprophylaxis therapy after discharge in this population requires further study.Venous thromboembolism is a leading cause of mortality among patients in hospital.^1,2 Major orthopedic surgery (e.g., hip or knee replacement) is associated with a high risk for postoperative venous thromboembolism.^1,3,4 Because the clinical diagnosis of venous thromboembolism is unreliable and its first manifestation may be a life-threatening pulmonary embolism,⁵ it is recommended that patients undergoing hip or knee replacement receive routine thromboprophylaxis with anticoagulant therapy after surgery unless they have contraindications to anticoagulant therapy.^1,3,5,6Thromboprophylaxis is commonly administered for the entire hospital stay, which is usually between 4 and 14 days.⁷ Expert consensus guidelines recommend that patients undergoing hip or knee replacement receive thromboprophylaxis medications for at least 10 days after surgery.⁶ These guidelines also recommend extended thromboprophylaxis for up to 28–35 days after surgery for patients undergoing hip replacement.⁶ Although there is evidence that extended thromboprophylaxis after hospital discharge is effective for reducing the risk of venous thromboembolism among patients who undergo hip replacement,⁸ the benefit among patients who undergo knee replacement has not been established.⁶ Thromboprophylaxis after discharge is likely to most benefit patients at high risk for venous thromboembolism, such as those with cancer, heart failure or major respiratory disease.^6–9 However, given that patients who undergo joint replacement are often elderly and have multiple comorbidities, the risks associated with extended thromboprophylaxis, particularly gastrointestinal bleeding and hemorrhagic strokes, may be substantial and may be relative contraindications for this therapy.¹⁰Among patients discharged home after hip-or knee-replacement surgery, we sought characterize the use of thromboprophylaxis after discharge and its consequences on risk of short-term mortality.     

Estimates of Water Ingestion for Women in Pregnant,Lactating, and Non-Pregnant and Non-Lactating Child-Bearing Age Groups Based on USDA's 1994–96, 1998 Continuing Survey of Food Intake by Individuals

Women in the child-bearing age of 15 to 44 years and, in particular, pregnant and lactating women in this age cohort are considered a sensitive subpopulation when assessing risk from ingestion of water because water borne contaminants may pose a risk not only to the mother but to the fetus or infant. This article presents estimates of daily average per capita water ingestion for women of child-bearing age and in three subgroups: pregnant, lactating, and non-pregnant/non-lactating women. Estimates of means and upper percentiles of subgroup ingestion distributions were generated using participant responses and survey weights from the United States Department of Agriculture's (USDA) 1994–96 and 1998 Continuing Survey of Food Intake by Individuals (CSFII). The ingestion estimates are empirical and not based on an assumed parametric distribution of daily average amount of water ingestion. Water occurring naturally in foods or added by manufacturers to commercial products is not included in the estimates presented. These estimates of water ingestion by women of child-bearing age are compared to those attributed to Ershow and Cantor (1989) Ershow, A G and Cantor, K P. 1989. Total Water and Tapwater Intake in the United States: Population-Based Estimates of Quantities and Sources Washington, DC, , USA National Cancer Institute Order #263-Md-810264 [Google Scholar] by Burmaster (1998) Burmaster, D E. 1998. Lognormal distributions of total water intake and tap water intake by pregnant and lactating women in the United States. Risk Anal, 18(2): 215–9. [Crossref] [Google Scholar]. These estimates, based on data collected in 1978, were used by Burmaster to characterize the distribution of daily average per capita ingestion as lognormal. The lognormal estimates of total water ingestion are generally greater than the total water ingestion estimates based on the CSFII data. Possible explanations for the differences are discussed.     

The emerging genetic architecture of type 2 diabetes

Type 2 diabetes is a genetically heterogeneous disease, with several relatively rare monogenic forms and a number of more common forms resulting from a complex interaction of genetic and environmental factors. Previous studies using a candidate gene approach, family linkage studies, and gene expression profiling uncovered a number of type 2 genes, but the genetic basis of common type 2 diabetes remained unknown. Recently, a new window has opened on defining potential type 2 diabetes genes through genome-wide SNP association studies of very large populations of individuals with diabetes. This review explores the pathway leading to discovery of these genetic effects, the impact of these genetic loci on diabetes risk, the potential mechanisms of action of the genes to alter glucose homeostasis, and the limitations of these studies in defining the role of genetics in this important disease.     

Miniaturization of scorpion beta-toxins uncovers a putative ancestral surface of interaction with voltage-gated sodium channels

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved betaalphabetabeta core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian beta-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated beta-toxins (DeltaDeltaBj-xtrIT and DeltaDeltaCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, DeltaDeltaBj-xtrIT and DeltaDeltaCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhalphaIT at insect sodium channels. Co-application of DeltaDeltaBj-xtrIT or DeltaDeltaCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at DeltaDeltaBj-xtrIT and DeltaDeltaCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.     

Loss of neuroprotective survival signal in mice lacking insulin receptor gene in rod photoreceptor cells

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rod-specific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to light-induced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection.