Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.     

Honeybee Associative Learning Performance and Metabolic Stress Resilience Are Positively Associated

Background

Social-environmental influences can affect animal cognition and health. Also, human socio-economic status is a covariate factor connecting psychometric test-performance (a measure of cognitive ability), educational achievement, lifetime health, and survival. The complimentary hypothesis, that mechanisms in physiology can explain some covariance between the same traits, is disputed. Possible mechanisms involve metabolic biology affecting integrity and stability of physiological systems during development and ageing. Knowledge of these relationships is incomplete, and underlying processes are challenging to reveal in people. Model animals, however, can provide insights into connections between metabolic biology and physiological stability that may aid efforts to reduce human health and longevity disparities.

Results

We document a positive correlation between a measure of associative learning performance and the metabolic stress resilience of honeybees. This relationship is independent of social factors, and may provide basic insights into how central nervous system (CNS) function and metabolic biology can be associated. Controlling for social environment, age, and learning motivation in each bee, we establish that learning in Pavlovian conditioning to an odour is positively correlated with individual survival time in hyperoxia. Hyperoxia induces oxidative metabolic damage, and provides a measure of metabolic stress resistance that is often related to overall lifespan in laboratory animals. The positive relationship between Pavlovian learning ability and stress resilience in the bee is not equally established in other model organisms so far, and contrasts with a genetic cost of improved associative learning found in Drosophila melanogaster.

Conclusions

Similarities in the performances of different animals need not reflect common functional principles. A correlation of honeybee Pavlovian learning and metabolic stress resilience, thereby, is not evidence of a shared biology that will give insight about systems integrity in people. Yet, the means to resolve difficult research questions often come from findings in distant areas of science while the model systems that turn out to be valuable are sometimes the least predictable. Our results add to recent findings indicating that honeybees can become instrumental to understanding how metabolic biology influences life outcomes.     

SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.     

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.     

A plague of waterfleas (<Emphasis Type="Italic">Bythotrephes</Emphasis>): impacts on microcrustacean community structure,seasonal biomass,and secondary production in a large inland-lake complex

The spiny cladoceran (Bythotrephes longimanus) is an invasive, predaceous zooplankter that is expanding from Great Lakes coastal waters into inland lakes within a northern latitudinal band. In a large, Boundary Water lake complex (largely within Voyageurs National Park), we use two comparisons, a 2-year spatial and a 12-year temporal, to quantify seasonal impacts on food webs and biomass, plus a preliminary calculation of secondary production decline. Bythotrephes alters the seasonal biomass pattern by severely depressing microcrustaceans during summer and early fall, when the predator is most abundant. Cladoceran and cyclopoid copepods suffer the most serious population declines, although the resistant cladoceran Holopedium is favored in spatial comparisons. Microcrustacean biomass is reduced 40–60 % and secondary production declines by about 67 %. The microcrustacean community shifts towards calanoid copepods. The decline in secondary production is due both to summer biomass loss and to the longer generation times of calanoid copepods (slower turnover). The Bythotrephes “top-down” perturbation appears to hold across small, intermediate, and large-sized lakes (i.e. appears scale-independent), and is pronounced when Bythotrephes densities reach 20–40 individuals L^?1. Induction tests with small cladocerans (Bosmina) suggest that certain native prey populations do not sense the exotic predator and are “blind-sided”. Failure of prey to deploy defenses could explain the disproportionate community impacts in New World versus Old World lakes.     

Genetic variation in gorillas

This review summarizes what is currently known concerning genetic variation in gorillas, on both inter- and intraspecific levels. Compared to the human species, gorillas, along with the other great apes, possess greater genetic variation as a consequence of a demographic history of rather constant population size. Data and hence conclusions from analysis of mitochondrial DNA (mtDNA), the usual means of describing intraspecific patterns of genetic diversity, are limited at this time. An important task for future studies is to determine the degree of confidence with which gorilla mtDNA can be analyzed, in view of the risk that one will inadvertently analyze artifactual rather than genuine sequences. The limited information available from sequences of nuclear genomic segments does not distinguish western from eastern gorillas, and, in comparison with results from the two chimpanzee species, suggests a relatively recent common ancestry for all gorillas. In the near future, the greatest insights are likely to come from studies aimed at genetic characterization of all individual members of social groups. Such studies, addressing topics such as behavior of individuals with kin and non-kin, and the actual success of male reproductive strategies, will provide a link between behavioral and genetic studies of gorillas.     

Research letter: Predictive value of a self-reported history of varicella infection in determining immunity in adults

THE RECENT INTRODUCTION OF A VACCINE FOR VARICELLA has raised questions about whether, for adults, a patient''s history of varicella infection is useful in determining if vaccination is necessary. We report findings on 184 family medicine patients aged 18 to 65 years who were asked if they had a history of varicella infection and were subsequently tested for varicella antibodies. A history of infection was positive for 114 (62%) of the participants and negative or uncertain for 70 (38%). All 114 subjects who reported a varicella infection history were immune. All 4 subjects who were not immune reported an uncertain or negative infection history. Except for people who are at increased risk of varicella infection or complications from infection, serologic testing may not be required for adults in the general population who have a history of varicella infection. Varicella infection (chickenpox) is highly contagious¹ and is spread by respiratory droplets or direct contact. Varicella can occur in nonimmune adults, in whom severity of the infection increases with age,² often causing serious morbidity and loss of work time. In Canada, 70% of 53 reported deaths caused by varicella between 1987 and 1996 occurred in adolescents and adults.³ When acquired during pregnancy, varicella can cause significant maternal, perinatal and infant morbidity.³ After varicella infection, more than 95% of people develop antibodies,⁴ which are detected by serologic testing and indicate lifelong immunity.The recent introduction of a vaccine for varicella means that nonimmune adults may benefit from vaccination.⁵ This has led to studies to determine whether a self-reported history of varicella infection is an accurate indication of the presence of antibodies. Studies to date have yielded disparate conclusions and recommendations,^{6,7,8,9,10,11} and no study has yet investigated this question for adults in a primary care population. Our objective was to determine, in an adult primary care population, the accuracy of a self-reported history of varicella infection in determining immunity.St. Michael''s Hospital Family Practice Unit serves a population with a wide range of socioeconomic groups and countries of origin. The mean patient age is 44 years, and 60% of patients are female. On randomly selected clinic dates between October and December 2000 we enrolled patients aged 18 to 65 years who were having blood taken for reasons other than serologic testing for varicella antibodies. Using a structured interview, patients were asked about their history of varicella infection (Box 1), and their blood samples were analyzed for antibody titres using the VZVscan latex agglutination test (Becton Dickinson, Cockeysville, Md.). We excluded patients with psychiatric or medical conditions impairing memory, a history of varicella vaccination or active varicella infection. All patients gave informed consent, and the study was approved by the St. Michael''s Hospital Research Ethics Board.Open in a separate windowBox 1A sample size of 200 patients provides power to detect a 95% confidence interval (CI) of ± 3% if the baseline positive predictive value of a history of varicella is 95%.Of 204 participants enrolled, 184 had a serologic test for varicella antibodies. For unknown reasons, the serologic test was not performed in the remaining 20 cases.Of the 184 participants whose blood sample was tested for varicella antibodies, 101 (55%) were women, 117 (64%) were born in North America, and 107 (58%) had postsecondary education. The mean age was 43 (standard deviation 12.8) years. Participants for whom serologic testing was not done had similar characteristics.All 114 (62%) of the subjects who reported a history of varicella infection were immune, for a positive predictive value of 100% (95% CI 97%–100%) (Open in a separate windowIn this study, a self-reported history of varicella infection was a highly accurate indicator of immunity to the pathogen. The 98% (180/184) seroprevalence of varicella–zoster virus antibodies in our study population was slightly higher than other population estimates,² and consequently a large majority of those who were uncertain of or reported no past infection were also immune.Although we recruited participants from only 1 family medicine clinic in an urban teaching centre, the patient population of the clinic is diverse in terms of patient country of origin, socioeconomic status and demographic characteristics.Whether particular vaccination strategies are appropriate or cost-effective depends on the population examined and their circumstances. The costs of serologic testing and vaccination and the potential financial, social and medical consequences of infection should be considered.For people who are at increased risk of varicella infection or for whom it is crucial to establish immunity, such as health care workers,^9,10,12,13 pregnant women^7,11,14 and household contacts of immunocompromised people, it may be prudent to have them undergo routine serologic testing regardless of their self-reported infection history.In this primary care setting, a positive history of varicella infection was an accurate indicator of the presence of antibodies. Except for people at high risk of varicella infection, serologic testing may not be required for adults in the general population who have had the infection. Vaccination should be offered to nonimmune patients.     

Death and renal transplantation among Aboriginal people undergoing dialysis

Background

Despite the increase in the number of Aboriginal people with end-stage renal disease around the world, little is known about their health outcomes when undergoing renal replacement therapy. We evaluated differences in survival and rate of renal transplantation among Aboriginal and white patients after initiation of dialysis.

Methods

Adult patients who were Aboriginal or white and who commenced dialysis in Alberta, Saskatchewan or Manitoba between Jan. 1, 1990, and Dec. 31, 2000, were recruited for the study and were followed until death, transplantation, loss to follow-up or the end of the study (Dec. 31, 2001). We used Cox proportional hazards models to examine the effect of race on patient survival and likelihood of transplant, with adjustment for potential confounders.

Results

Of the 4333 adults who commenced dialysis during the study period, 15.8% were Aboriginal and 72.4% were white. Unadjusted rates of death per 1000 patient-years during the study period were 158 (95% confidence interval [CI] 144–176) for Aboriginal patients and 146 (95% CI 139–153) for white patients. When follow-up was censored at the time of transplantation, the age-adjusted risk of death after initiation of dialysis was significantly higher among Aboriginal patients than among white patients (hazard ratio [HR] 1.15, 95% CI 1.02–1.30). The greater risk of death associated with Aboriginal race was no longer observed after adjustment for diabetes mellitus and other comorbid conditions (adjusted HR 0.89, 95% CI 0.77–1.02) and did not appear to be associated with socioeconomic status. During the study period, unadjusted transplantation rates per 1000 patient-years were 62 (95% CI 52–75) for Aboriginal patients and 133 (95% CI 125–142) for white patients. Aboriginal patients were significantly less likely to receive a renal transplant after commencing dialysis, even after adjustment for potential confounders (HR 0.43, 95% CI 0.35–0.53). In an additional analysis that included follow-up after transplantation for those who received renal allografts, the age-adjusted risk of death associated with Aboriginal race (HR 1.36, 95% CI 1.21–1.52) was higher than when follow-up after transplantation was not considered, perhaps because of the lower rate of transplantation among Aboriginals.

Interpretation

Survival among dialysis patients was similar for Aboriginal and white patients after adjustment for comorbidity. However, despite universal access to health care, Aboriginal people had a significantly lower rate of renal transplantation, which might have adversely affected their survival when receiving renal replacement therapy.In North America and the Antipodes, the incidence of diabetes among adolescent and adult Aboriginals has risen dramatically,^1,2,3,4 with corresponding increases in the prevalence of diabetic nephropathy.^5,6,7 Aboriginal people in Canada have experienced disproportionately high incidence rates of end-stage renal disease (ESRD), with an 8-fold increase in the number of prevalent dialysis patients between 1980 and 2000.⁸ Although the incidence of ESRD appears to have decreased in recent years, the prevalence of diabetes mellitus and its complications are rising, especially among young people.^9,10,11Most work evaluating health outcomes among Aboriginal people considers either the general population¹²or diseases for which interventions are implemented over a short period, such as alcohol abuse,¹³ injury¹⁴ or critical illness.¹⁵ Death and markers of poor health are significantly more common among Aboriginal people than among North Americans of European ancestry, perhaps because of the greater prevalence of diabetes mellitus, adverse health effects due to lower socioeconomic status¹⁶ and reduced access to primary care.¹⁷ Aboriginal patients may also face unique barriers to care, including mistrust of non-Aboriginal providers, institutional discrimination or preference for traditional remedies.¹⁸ These factors may be most relevant when contact with physicians is infrequent, which obstructs development of a therapeutic relationship. In contrast, ESRD is a chronic illness that requires ongoing care from a relatively small, stable multidisciplinary team.Although recent evidence highlights racial inequalities in morbidity and mortality among North Americans with ESRD, most studies have focused on black or Hispanic populations.¹⁹We conducted this study to evaluate rates of death and renal transplantation among Aboriginal people after initiation of dialysis in Alberta, Saskatchewan and Manitoba.