Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites

Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structur

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region

Non-specific low back pain in primary care in the Spanish National Health Service: a prospective study on clinical outcomes and determinants of management

BACKGROUND: The Spanish National Health Service is a universal and free health care system. Non-specific low back pain (LBP) is a prevalent disorder, generating large health and social costs. The objectives of this study were to describe its management in primary care, to assess patient characteristics that influence physicians' decisions, and to describe clinical outcome at 2 months. METHODS: A cross-sectional sample of 648 patients with non-specific low back pain was recruited by 75 physicians (out of 361 – 20.8%) working in 40 primary care centers in 10 of the 17 administrative regions in Spain, covering 693,026 out of the 40,499,792 inhabitants. Patients were assessed on the day they were recruited, and prospectively followed-up 14 and 60 days later. The principal patient characteristics that were analyzed were: sex, duration of the episode, history of LBP, working status, severity of LBP, leg pain and disability, and results of straight leg raising test. Descriptors of management were: performance of the straight leg raising test, ordering of diagnostic procedures, prescription of drug treatment, referral to physical therapy, rehabilitation or surgery, and granting of sick leave. Regression analysis was used to analyze the relationship between patients' baseline characteristics and physicians' management decisions. Only workers were included in the models on sick leave. RESULTS: Mean age (SD) of included patients was 46.5 (15.5) years, 367 (56.6%) were workers, and 338 (52.5%) were females. Median (25th–75th interquartile range) duration of pain when entering the study was 4 (2–10) days and only 28 patients (4.3%) had chronic low back pain. Diagnostic studies included plain radiographs in 43.1% of patients and CT or MRI scans in 18.8%. Drug medication was prescribed to 91.7% of patients, 19.1% were sent to physical therapy or rehabilitation, and 9.6% were referred to surgery. The main determinants of the clinical management were duration of the episode and, to a lesser extent, the intensity of the pain (especially leg pain), a positive straight leg raising test, and degree of disability. The main determinant of sick leave was the degree of disability, followed by the characteristics of the labor contract and the intensity of leg pain (but not low back pain). After at least 2 months of treatment, 37% of patients were still in pain and approximately 10% of patients had not improved or had worsened. CONCLUSION: Although the use of X-Rays is high, determinants of physicians' management of LBP in primary care made clinical sense and were consistent with patterns suggested by evidence-based recommendations. However, after 2 months of treatment more than one third of patients continued to have back pain and about 10% had worsened

Need for enforcement of ethicolegal education – an analysis of the survey of postgraduate clinical trainees

BACKGROUND: The number of medical lawsuits in Japan was between 14 and 21 each year before 1998, but increased to 24 to 35 per year after 1999. There were 210 lawsuits during this 10-year period. There is a need for skills and knowledge related to ethics, which is as fundamental to the practice of medicine as basic sciences or clinical skills. in Japan education in ethics is relatively rare and its importance is not yet recognized. Establishing ethics education using legal precedents, which has already been achieved in Western countries, will be a very important issue in Japan. In the present study, a questionnaire survey was conducted among graduate intern doctors, in order to investigate whether ethics education using precedents might have a positive effect in Japan. METHODS: In 2002, a questionnaire survey entitled Physicians' Clinical Ethics was carried out in a compulsory orientation lecture given to trainees before they started clinical practice in our hospital. The attendees at this lecture were trainees who came from colleges in various districts of Japan. During the lecture, 102 questionnaires were distributed, completed by attendees and collected. The recovery rate was 100%. The questionnaire consisted of 22 questions (in three categories), of which 20 were answered by multiple choices, and the other two were answered by description. The time required to complete the questionnaire was about 10 minutes. RESULTS: The recovered questionnaires were analyzed using statistical analysis software (SPSS for Windows, Release 10.07J-1/June/2000), in addition to simple statistical analysis. answers using multiple choices for the 20 questions in the questionnaire were input into SPSS. The principal component analysis was performed for each question. As a result, the item that came to the fore was "legal precedent". Since many intern doctors were interested in understanding laws and precedents, learning about ethical considerations through education using precedents might better meet with their needs and interests. CONCLUSION: We applied a new method in which the results of principal component analysis and frequencies of answers to other questions were combined. From this we deduced that the precedent education used in Western countries was useful to help doctors acquire ethical sensitivity and was not against their will. A relationship was found between reading precedents and the influence of lawsuits, and it was thought that student participation-type precedent education would be useful for doctors in order to acquire ethical sensitivity

A model of impairment and functional limitation in rheumatoid arthritis

BACKGROUND: We have previously proposed a theoretical model for studying physical disability and other outcomes in rheumatoid arthritis (RA). The purpose of this paper is to test a model of impairment and functional limitation in (RA), using empirical data from a sample of RA patients. We based the model on the disablement process framework. METHODS: We posited two distinct types of impairment in RA: 1) Joint inflammation, measured by the tender, painful and swollen joint counts; and 2) Joint deformity, measured by the deformed joint count. We hypothesized direct paths from the two impairments to functional limitation, measured by the shirt-button speed, grip strength and walking velocity. We used structural equation modeling to test the hypothetical relationships, using empirical data from a sample of RA patients recruited from six rheumatology clinics. RESULTS: The RA sample was comprised of 779 RA patients. In the structural equation model, the joint inflammation impairment displayed a strong significant path toward the measured variables of joint pain, tenderness and swelling (standardized regression coefficients 0.758, 0.872 and 0.512, P ≤ 0.001 for each). The joint deformity impairment likewise displayed significant paths toward the measured upper limb, lower limb, and other deformed joint counts (standardized regression coefficients 0.849, 0.785, 0.308, P ≤ 0.001 for each). Both the joint inflammation and joint deformity impairments displayed strong direct paths toward functional limitation (standardized regression coefficients of -0.576 and -0.564, respectively, P ≤ 0.001 for each), and explained 65% of its variance. Model fit to data was fair to good, as evidenced by a comparative fit index of 0.975, and the root mean square error of approximation = 0.058. CONCLUSION: This evidence supports the occurrence of two distinct impairments in RA, joint inflammation and joint deformity, that together, contribute strongly to functional limitations in this disease. These findings may have implications for investigators aiming to measure outcome in RA

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Load and speed effects on the cervical flexion relaxation phenomenon

<p>Abstract</p> <p>Background</p> <p>The flexion relaxation phenomenon (FRP) represents a well-studied neuromuscular response that occurs in the lumbar and cervical spine. However, the cervical spine FRP has not been investigated extensively, and the speed of movement and loading effects remains to be characterized. The objectives of the present study were to evaluate the influence of load and speed on cervical FRP electromyographic (EMG) and kinematic parameters and to assess the measurement of cervical FRP kinematic and EMG parameter repeatability.</p> <p>Methods</p> <p>Eighteen healthy adults (6 women and 12 men), aged 20 to 39 years, participated in this study. They undertook 2 sessions in which they had to perform a standardized cervical flexion/extension movement in 3 phases: complete cervical flexion; the static period in complete cervical flexion; and extension with return to the initial position. Two different rhythm conditions and 3 different loading conditions were applied to assess load and speed effects. Kinematic and EMG data were collected, and dependent variables included angles corresponding to the onset and cessation of myoelectric silence as well as the root mean square (RMS) values of EMG signals. Repeatability was examined in the first session and between the 2 sessions.</p> <p>Results</p> <p>Statistical analyses revealed a significant load effect (P < 0.001). An augmented load led to increased FRP onset and cessation angles. No load × speed interaction effect was detected in the kinematics data. A significant load effect (P < 0.001) was observed on RMS values in all phases of movement, while a significant speed effect (P < 0.001) could be seen only during the extension phase. Load × speed interaction effect was noted in the extension phase, where higher loads and faster rhythm generated significantly greater muscle activation. Intra-session and inter-session repeatability was good for the EMG and kinematic parameters.</p> <p>Conclusions</p> <p>The load increase evoked augmented FRP onset and cessation angles as well as heightened muscle activation. Such increments may reflect the need to enhance spinal stability under loading conditions. The kinematic and EMG parameters showed promising repeatability. Further studies are needed to assess kinematic and EMG differences between healthy subjects and patients with neck pain.</p

Relationships of the Location and Content of Rounds to Specialty, Institution, Patient-Census, and Team Size

OBJECTIVE: Existing observational data describing rounds in teaching hospitals are 15 years old, predate duty-hour regulations, are limited to one institution, and do not include pediatrics. We sought to evaluate the effect of medical specialty, institution, patient-census, and team participants upon time at the bedside and education occurring on rounds. METHODS AND PARTICIPANTS: Between December of 2007 and October of 2008 we performed 51 observations at Lucile Packard Children's Hospital, Seattle Children's Hospital, Stanford University Hospital, and the University of Washington Medical Center of 35 attending physicians. We recorded minutes spent on rounds in three location and seven activity categories, members of the care team, and patient-census. RESULTS: Results presented are means. Pediatric rounds had more participants (8.2 vs. 4.1 physicians, p<.001; 11.9 vs. 2.4 non-physicians, p<.001) who spent more minutes in hallways (96.9 min vs. 35.2 min, p<.001), fewer minutes at the bedside (14.6 vs. 38.2 min, p = .01) than internal medicine rounds. Multivariate regression modeling revealed that minutes at the bedside per patient was negatively associated with pediatrics (-2.77 adjusted bedside minutes; 95% CI -4.61 to -0.93; p<.001) but positively associated with the number of non-physician participants (0.12 adjusted bedside minutes per non physician participant; 95% CI 0.07 to 0.17; p = <.001). Education minutes on rounds was positively associated with the presence of an attending physician (2.70 adjusted education minutes; 95% CI 1.27 to 4.12; p<.001) and with one institution (1.39 adjusted education minutes; 95% CI 0.26 to 2.53; p = .02). CONCLUSIONS: Pediatricians spent less time at the bedside on rounds than internal medicine physicians due to reasons other than patient-census or the number of participants in rounds. Compared to historical data, internal medicine rounds were spent more at the bedside engaged in patient care and communication, and less upon educational activities

Polymorphisms of Serotonin Receptor 2A and 2C Genes and COMT in Relation to Obesity and Type 2 Diabetes

BACKGROUND:Candidate genes of psychological importance include 5HT2A, 5HT2C, and COMT, implicated in the serotonin, noradrenaline and dopamine pathways, which also may be involved in regulation of energy balance. We investigated the associations of single nucleotide polymorphisms (SNPs) of these genes with obesity and metabolic traits. METHODOLOGY/PRINCIPAL FINDINGS:In a population of 166 200 young men examined at the draft boards, obese men (n = 726, BMI> or =31.0 kg/m(2)) and a randomly selected group (n = 831) were re-examined at two surveys at mean ages 46 and 49 years (S-46, S-49). Anthropometric, physiological and biochemical measures were available. Logistic regression analyses were used to assess age-adjusted odds ratios. No significant associations were observed of 5HT2A rs6311, 5HT2C rs3813929 and COMT rs4680 with obesity, except that COMT rs4680 GG-genotype was associated with fat-BMI (OR = 1.08, CI = 1.01-1.16). The SNPs were associated with a number of physiological variables; most importantly 5HT2C rs3813929 T-allele was associated with glucose (OR = 4.56, CI = 1.13-18.4) and acute insulin response (OR = 0.65, CI = 0.44-0.94) in S-49. COMT rs4680 GG-genotype was associated with glucose (OR = 1.04, CI = 1.00-1.09). Except for an association between 5HT2A rs6311 and total-cholesterol at both surveys, significant in S-46 (OR = 2.66, CI = 1.11-6.40), no significant associations were observed for the other phenotypes. Significant associations were obtained when combined genotype of 5HT2C rs3813929 and COMT rs4680 were examined in relation to BMI (OR = 1.12, CI = 1.03-1.21), fat-BMI (OR = 1.22, CI = 1.08-1.38), waist (OR = 1.13, CI = 1.04-1.22), and cholesterol (OR = 5.60, CI = 0.99-31.4). Analyses of impaired glucose tolerance (IGT) and type 2 diabetes (T2D) revealed, a 12.3% increased frequency of 5HT2C rs3813929 T-allele and an 11.6% increased frequency of COMT rs4680 GG-genotype in individuals with IGT or T2D (chi(2), p = 0.05 and p = 0.06, respectively). Examination of the combined genotypes of 5HT2C and COMT showed a 34.0% increased frequency of IGT or T2D (chi(2), p = 0.01). CONCLUSIONS:The findings lend further support to the involvement of serotonin, noradrenaline and dopamine pathways on obesity and glucose homeostasis, in particular when combined genotype associations are explored

The Combined Effects of Amino Acid Substitutions and Indels on the Evolution of Structure within Protein Families

BACKGROUND: In the process of protein evolution, sequence variations within protein families can cause changes in protein structures and functions. However, structures tend to be more conserved than sequences and functions. This leads to an intriguing question: what is the evolutionary mechanism by which sequence variations produce structural changes? To investigate this question, we focused on the most common types of sequence variations: amino acid substitutions and insertions/deletions (indels). Here their combined effects on protein structure evolution within protein families are studied. RESULTS: Sequence-structure correlation analysis on 75 homologous structure families (from SCOP) that contain 20 or more non-redundant structures shows that in most of these families there is, statistically, a bilinear correlation between the amount of substitutions and indels versus the degree of structure variations. Bilinear regression of percent sequence non-identity (PNI) and standardized number of gaps (SNG) versus RMSD was performed. The coefficients from the regression analysis could be used to estimate the structure changes caused by each unit of substitution (structural substitution sensitivity, SSS) and by each unit of indel (structural indel sensitivity, SIDS). An analysis on 52 families with high bilinear fitting multiple correlation coefficients and statistically significant regression coefficients showed that SSS is mainly constrained by disulfide bonds, which almost have no effects on SIDS. CONCLUSIONS: Structural changes in homologous protein families could be rationally explained by a bilinear model combining amino acid substitutions and indels. These results may further improve our understanding of the evolutionary mechanisms of protein structures