Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers

UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 ? resolution, which shows nsp10 bound to nsp16 through a ～930 ?2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.     

The length weight relationship, age, growth and reproduction of the roach Rutilus rutilus (L.) in Lake Volvi

The length-weight relationship of a sample of 233 roach ( Rutilus rutilus ) can be described by the following equations: y =0·0356 x ^3·405 and y =0·0215 ×^3·606 for males and females respectively. In both equations y equals the body weight in grams and × is the standard length in centimetres. The average condition factor K was 2·01 with a range of 1·71 to 2·26. The roach's span of life was 13 years for both sexes. The growth increment is greater during the first year of life (about 56 mm), decreasing to approximately 17 mm at the end of the sixth year of life and then becoming constant at about 12 mm per year. Roach become sexually mature at age 1 + for males and one year later for females. The mean absolute fecundity was 9294 eggs; with a range from 920 to 32 810. The growth of the gonads is related to the age of the fish. Spawning occurs during the first half of April, at a mean water temperature of 10° C.     

Types of supernumerary outgrowths produced after inverting the dorsoventral limb axis of the anuranBufo bufo

Summary Contralateral grafts were performed on the larval limb buds of the anuranBufo bufo. The dorsoventral axis of 80 buds at stages IV or V was inverted. Ten tadpoles were used as controls. Fifty-two supernumerary structures developed, all of them in dorsal or ventral locations on the host stump. The majority (32 out of the 44 outgrowths with more than 3 toes) were normal limbs of stump handedness. However, the following abnormal structures were also observed: 2 double-posterior, 3 mixed-symmetric, and 7 undetermined cases. These results are in agreement with the predictions of a hierarchical polar coordinate model for epimorphic regeneration.     

Outcome of low back pain in general practice: a prospective study

Objectives: To investigate the claim that 90% of episodes of low back pain that present to general practice have resolved within one month. Design: Prospective study of all adults consulting in general practice because of low back pain over 12 months with follow up at 1 week, 3 months, and 12 months after consultation. Setting: Two general practices in south Manchester. Subjects: 490 subjects (203 men, 287 women) aged 18-75 years. Main outcome measures: Proportion of patients who have ceased to consult with low back pain after 3 months; proportion of patients who are free of pain and back related disability at 3 and 12 months. Results: Annual cumulative consultation rate among adults in the practices was 6.4%. Of the 463 patients who consulted with a new episode of low back pain, 275 (59%) had only a single consultation, and 150 (32%) had repeat consultations confined to the 3 months after initial consultation. However, of those interviewed at 3 and 12 months follow up, only 39/188 (21%) and 42/170 (25%) respectively had completely recovered in terms of pain and disability. Conclusions: The results are consistent with the interpretation that 90% of patients with low back pain in primary care will have stopped consulting with symptoms within three months. However most will still be experiencing low back pain and related disability one year after consultation.

Key messages

• It is widely believed that 90% of episodes of low back pain seen in general practice resolve within one month
• In a large population based study we examined the outcome of episodes of low back pain in general practice with respect to both consultation behaviour and self reported pain and disability
• While 90% of subjects consulting general practice with low back pain ceased to consult about the symptoms within three months, most still had substantial low back pain and related disability
• Only 25% of the patients who consulted about low back pain had fully recovered 12 months later
• Since most consulters continue to have long term low back pain and disability, effective early treatment could reduce the burden of these symptoms and their social, economic, and medical impact

     

Glucose analogue inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes

The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analogue inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for additional atoms attached at the C1-beta position of glucose and some scope for additional atoms at the C1-alpha position. Kinetic parameters were determined for alpha-D-glucose: Ki = 1.7 mM, Hill coefficient n = 1.5, and alpha (synergism with caffeine) = 0.2. For beta-D-glucose, Ki = 7.4 mM, n = 1.5, and alpha = 0.4. More than 20 glucose analogues have been synthesized and tested in kinetic experiments. Most were less effective inhibitors than glucose itself and the best inhibitor was alpha-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, alpha = 0.4). The binding of 14 glucose analogues to glycogen phosphorylase b in the crystal has been studied at 2.4-A resolution and the structure have been refined to crystallographic R values of less than 0.20. The kinetic and crystallographic studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogues. The results show the discrimination against beta-D-glucose in favor of alpha-D-glucose is achieved by an additional hydrogen bond made in the alpha-glucose complex through water to a protein group and an unfavorable environment for a polar group in the beta pocket. The compound alpha-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogues with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl beta-D-glucoside) with those whose substituent atoms are more rigid (e.g., beta-azidomethyl-1-deoxyglucose or beta-cyanomethyl-1-deoxyglucose) indicates that although all three compounds make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, alpha-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compound made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a number of compounds (beta-aminoethyl, beta-mesylate, and beta-azidomethyl analogues). The beta pocket was shown to bind gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.     

Eugene I. Rabinowitch: A prophet of photosynthesis and of peace in the world

Photosynthesis Research - More than 45 years have passed since Eugene I. Rabinowitch died, on May 15, 1973, at the age of 75, but many still remember him as a photosynthesis giant, the...     

Mediational Effects of Self-Efficacy Dimensions in the Relationship between Knowledge of Dengue and Dengue Preventive Behaviour with Respect to Control of Dengue Outbreaks: A Structural Equation Model of a Cross-Sectional Survey

Background

Dengue fever is endemic in Malaysia, with frequent major outbreaks in urban areas. The major control strategy relies on health promotional campaigns aimed at encouraging people to reduce mosquito breeding sites close to people''s homes. However, such campaigns have not always been 100% effective. The concept of self-efficacy is an area of increasing research interest in understanding how health promotion can be most effective. This paper reports on a study of the impact of self-efficacy on dengue knowledge and dengue preventive behaviour.

Methods and Findings

We recruited 280 adults from 27 post-outbreak villages in the state of Terengganu, east coast of Malaysia. Measures of health promotion and educational intervention activities and types of communication during outbreak, level of dengue knowledge, level and strength of self-efficacy and dengue preventive behaviour were obtained via face-to-face interviews and questionnaires. A structural equation model was tested and fitted the data well (χ² = 71.659, df = 40, p = 0.002, RMSEA = 0.053, CFI = 0.973, TLI = 0.963). Mass media, local contact and direct information-giving sessions significantly predicted level of knowledge of dengue. Level and strength of self-efficacy fully mediated the relationship between knowledge of dengue and dengue preventive behaviours. Strength of self-efficacy acted as partial mediator in the relationship between knowledge of dengue and dengue preventive behaviours.

Conclusions

To control and prevent dengue outbreaks by behavioural measures, health promotion and educational interventions during outbreaks should now focus on those approaches that are most likely to increase the level and strength of self-efficacy.