Frequency and Distribution of Refractive Error in Adult Life: Methodology and Findings of the UK Biobank Study

PurposeTo report the methodology and findings of a large scale investigation of burden and distribution of refractive error, from a contemporary and ethnically diverse study of health and disease in adults, in the UK.MethodsU K Biobank, a unique contemporary resource for the study of health and disease, recruited more than half a million people aged 40–69 years. A subsample of 107,452 subjects undertook an enhanced ophthalmic examination which provided autorefraction data (a measure of refractive error). Refractive error status was categorised using the mean spherical equivalent refraction measure. Information on socio-demographic factors (age, gender, ethnicity, educational qualifications and accommodation tenure) was reported at the time of recruitment by questionnaire and face-to-face interview.ResultsFifty four percent of participants aged 40–69 years had refractive error. Specifically 27% had myopia (4% high myopia), which was more common amongst younger people, those of higher socio-economic status, higher educational attainment, or of White or Chinese ethnicity. The frequency of hypermetropia increased with age (7% at 40–44 years increasing to 46% at 65–69 years), was higher in women and its severity was associated with ethnicity (moderate or high hypermetropia at least 30% less likely in non-White ethnic groups compared to White).ConclusionsRefractive error is a significant public health issue for the UK and this study provides contemporary data on adults for planning services, health economic modelling and monitoring of secular trends. Further investigation of risk factors is necessary to inform strategies for prevention. There is scope to do this through the planned longitudinal extension of the UK Biobank study.     

Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5.

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.     

Fatigue after stroke: baseline predictors and influence on survival. Analysis of data from UK patients recruited in the International Stroke Trial

Background and Purpose

Little is known about the associations of post-stroke fatigue or its influence on survival. The vitality component of the Short Form 36 (SF-36) is a valid and reliable measure of post-stroke fatigue. We sought to identify associates of post-stroke fatigue and determine whether fatigue predicted survival.

Methods

We used SF-36 vitality scores obtained by postal questionnaires from 1080 UK patients randomised in the International Stroke Trial, at a mean of 64 weeks after stroke onset. We used logistic regression to explore factors at randomisation which predicted SF-36 vitality at follow-up, and the relationship between SF-36 vitality and both SF-36 mental health and SF-36 emotional role function at follow-up. We used Cox proportional hazards to explore the influence of SF-36 vitality at follow-up on subsequent survival, using four different statistical models for handling missing data.

Results

Female sex, increasing age, lower mental health and lower emotional role function scores were associated with greater degrees of fatigue after stroke (i.e. lower vitality scores) but these factors explained <30% of the variance (R²) in fatigue. In two models, fatigue at follow-up was associated with shorter subsequent survival.

Conclusion

Increasing age, female sex, emotional role function and mental health were associated with increased fatigue at a mean of 64 weeks after stroke onset, but explained less than 30% of the variance. Fatigue was associated with reduced subsequent long-term survival in 2/4 models. Further work is needed to identify the biological substrate of fatigue and to clarify its influence on survival.     

Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase

UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.     

Characterization of protein and transcript levels of the chaperonin containing tailless complex protein-1 and tubulin during light-regulated growth of oat seedlings

In grass seedlings the network of cortical microtubules is reorganized during light-dependent growth of coleoptiles and mesocotyls. We investigated the effects of light-dependent growth on the relative steady-state levels of the mRNAs and protein levels of alpha-tubulin and the epsilon-subunit of the chaperonin containing tailless complex protein-1 in oat (Avena sativa) coleoptiles, which were grown in different light conditions to establish different growth responses. The soluble pools of the epsilon-subunit of the chaperonin containing tailless complex protein-1 and alpha-tubulin decreased in nonelongating coleoptiles, suggesting that the dynamics of the light-regulated soluble pool reflect the processes occurring during reorganization of cortical microtubules. The shifts in pool sizes are discussed in relation to the machinery that controls the dynamic structure of cortical microtubules in plant cells.     

Proteinase yscD. Purification and characterization of a new yeast peptidase

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.     

Applications of neutron activation analysis to the study of age-related neurological diseases

Although the etiology and pathogenesis of Alzheimer’s disease, Pick’s disease, and amyotrophic lateral sclerosis are still unknown, it has been suggested that perturbations in element metabolism may play a role. Even if not causative factors, these imbalances may prove to be markers that could aid in diagnosis. We have employed a sequential neutron activation analysis (NAA) procedure to determine elemental concentrations in brain, hair, fingernails, blood, and cerebrospinal fluid (CSF) of these patients and age-matched controls. Samples are first irradiated with accelerator-produced 14-MeV neutrons for determination of nitrogen and phosphorus, then with reactor thermal neutrons for the instrumental determination of 16–18 minor and trace elements, and, finally, reactor-irradiated again, followed by a rapid radiochemical separation procedure (RNAA) to determine four additional elements. Major advantages of NAA are: (1) its simultaneous multielement capability; (2) the relative freedom from reagent and laboratory contamination; (3) the absence of major matrix effects; and (4) an adequate sensitivity for most elements of interest. Ranges of concentrations by INAA and RNAA in selected control tissues and interelement correlations in control brain are presented to illustrate results obtained by the procedure. Longitudinal studies of tissues from Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) patients are still in progress.     

Expression of three genes coding for 135-kilodalton entomocidal proteins inBacillus thuringiensis kurstaki

The HD-1 strain ofBacillus thuringiensis (B.t.)kurstaki contains three homologous genes coding for 130–134-kilodalton entomocidal proteins [13]. In the present study, expression levels of these genes in strains of B.t.kurstaki were determined. In attempts to isolate a protein coded by a single gene, a number of variants were derived from strains of B.t.kurstaki, such as HD-263 and HD-1, by plasmid curing. The entomocidal proteins produced by the parental strains and their plasmid-cured variants were isolated by Sephacryl S-300 column chromatography and peptide-mapped by high performance liquid chromatography (HPLC). The results indicated that HD-263 produced two distinctive proteins, one identical with the protein of HD-73, which contains only a 6.6 kb Hind III class gene, and the other protein presumably coded by a 4.5 kb Hind III class gene. HPLC analysis revealed that 70% of the total protein in the HD-263 crystals consisted of the product of the 6.6 kb gene (6.6-kb protein), and the remaining 30% was the 4.5-kb protein. In the case of HD-1, the crystal consisted of at least two different proteins in equal amounts (50% each). The gene coding for one of these proteins was presumed to be a 5.3 kb Hind III class gene. The remaining 50% of the HD-1 crystal was accounted for by a protein similar to the 4.5-kb protein identified in HD-263. It appeared that the 6.6-kb protein was expressed poorly, if it was indeed expressed, in the HD-1 strain.     

Feeder cells impart a growth stimulus to recipient epithelial cells by direct contact.

Mouse mammary epithelial cells have been shown to proliferate when cultured in the same vessel with lethally irradiated cells of the LA7 rat mammary tumor line. Presented here are experiments that indicate that the LA7 feeder cells stimulate growth of the normal mouse mammary cells by a mechanism that involves direct contact between the two cell types. It is possible that the LA7 feeder cells stimulate proliferation by secretion of a labile growth factor, by secretion of a soluble growth factor in such low concentrations that dilution by travel over a distance makes it less effective, that the stimulus is transduced directly through membrane receptors on the recipient epithelial cells, or that a growth message is sent through gap junctions between cells. This feeder cell system is proposed as an in vitro model for epithelial wound healing.