Thiamine and magnesium deficiencies: keys to disease.

Thiamine deficiency (TD) is accepted as the cause of beriberi because of its action in the metabolism of simple carbohydrates, mainly as the rate limiting cofactor for the dehydrogenases of pyruvate and alpha-ketoglutarate, both being critical to the action of the citric acid cycle. Transketolase, dependent on thiamine and magnesium, occurs twice in the oxidative pentose pathway, important in production of reducing equivalents. Thiamine is also a cofactor in the dehydrogenase complex in the degradation of the branched chain amino acids, leucine, isoleucine and valine. In spite of these well accepted facts, the overall clinical effects of TD are still poorly understood. Because of the discovery of 2-hydroxyacyl-CoA lyase (HACL1) as the first peroxisomal enzyme in mammals found to be dependent on thiamine pyrophosphate (TPP) and the ability of thiamine to bind with prion protein, these factors should improve our clinical approach to TD. HACL1 has two important roles in alpha oxidation, the degradation of phytanic acid and shortening of 2-hydroxy long-chain fatty acids so that they can be degraded further by beta oxidation. The downstream effects of a lack of efficiency in this enzyme would be expected to be critical in normal brain metabolism. Although TD has been shown experimentally to produce reversible damage to mitochondria and there are many other causes of mitochondrial dysfunction, finding TD as the potential biochemical lesion would help in differential diagnosis. Stresses imposed by infection, head injury or inoculation can initiate intermittent cerebellar ataxia in thiamine deficiency/dependency. Medication or vaccine reactions appear to be more easily initiated in the more intelligent individuals when asymptomatic marginal malnutrition exists. Erythrocyte transketolase testing has shown that thiamine deficiency is widespread. It is hypothesized that the massive consumption of empty calories, particularly those derived from carbohydrate and fat, results in a high calorie/thiamine ratio as a major cause of disease. Because mild to moderate TD results in pseudo hypoxia in the limbic system and brainstem, emotional and stress reflexes of the autonomic nervous system are stimulated and exaggerated, producing symptoms often diagnosed as psychosomatic disease. If the biochemical lesion is recognized at this stage, the symptoms are easily reversible. If not, and the malnutrition continues, neurodegeneration follows and results in a variety of chronic brain diseases. Results from acceptance of the hypothesis could be tested by performing erythrocyte transketolase tests to pick out those with TD and supplementing the affected individuals with the appropriate dietary supplements.

Bridging the gap: an integrated paediatric to adult clinical service for young adults with kidney failure.

PROBLEM: Transition from paediatric to adult care of young adults with chronic diseases is poorly coordinated, often delayed, and usually managed through a single referral letter. About 35% of young adults lose a successfully functioning kidney transplant within 36 months of transfer from paediatric to adult services. DESIGN: Before and after study of the impact of a new integrated paediatric-adult clinical service for patients with kidney failure. SETTING: Adult renal centre in Oxford and two paediatric renal centres in London. STRATEGIES FOR CHANGE: An integrated paediatric-young adult joint transition clinic and care pathway was established in 2006, in conjunction with a young adult clinical service with regular community based clinics. Previously, young adult transplant recipients were transferred by a single referral letter to an adult renal consultant and managed in a conventional adult clinic. KEY MEASURES FOR IMPROVEMENT: Rates of acute rejection and loss of kidney transplants five years before and five years after the introduction of the integrated young adult care pathway. EFFECTS OF THE CHANGE: Nine young adult kidney transplant recipients were transferred directly to adult care between 2000 and 2006 (group 1). From 2006 to 2010, 12 young adult transplant recipients underwent integrated transition into the new young adult service (group 2). Six transplants were lost in group 1 (67%) compared with no transplant losses in group 2. LESSONS LEARNT: Implementing an integrated transition clinic, coupled with improving young adults' healthcare experience through a young adult clinic, improved patient adherence to regular medication and engagement with healthcare providers, as judged by reduced transplant failure rates. This model may be applicable to other young adult populations with chronic disease transferring to adult healthcare.

A screen-printed, amperometric biosensor array incorporated into a novel automated system for the simultaneous determination of organophosphate pesticides.

Organophosphate pesticides present serious risks to human and environmental health. A rapid reliable, economical and portable analytical system will be of great benefit in the detection and prevention of contamination. A biosensor array based on six acetylcholinesterase enzymes for use in a novel automated instrument incorporating a neural network program is described. Electrochemical analysis was carried out using chronoamperometry and the measurement was taken 10s after applying a potential of 0 V vs. Ag/AgCl. The total analysis time for the complete assay was less than 6 min. The array was used to produce calibration data with six organophosphate pesticides (OPs) in the concentration range of 10(-5) M to 10(-9) M to train a neural network. The output of the neural network was subsequently evaluated using different sample matrices. There were no detrimental matrix effects observed from water, phosphate buffer, food or vegetable extracts. Furthermore, the sensor system was not detrimentally affected by the contents of water samples taken from each stage of the water treatment process. The biosensor system successfully identified and quantified all samples where an OP was present in water, food and vegetable extracts containing different OPs. There were no false positives or false negatives observed during the evaluation of the analytical system. The biosensor arrays and automated instrument were evaluated in situ in field experiments where the instrument was successfully applied to the analysis of a range of environmental samples. It is envisaged that the analytical system could provide a rapid detection system for the early warning of contamination in water and food.

Abiotic variables effect differential expression of latent infections in beech (Fagus sylvatica).

• Effects of temperature and gaseous regimes on development of fungal communities, from latent infections in twigs, branches and stems of European beech ( Fagus sylvatica ), were investigated through controlled drying. • Hypoxylon fragiforme was identified as a latent invader within the xylem of all orders of host stem and branch material. Explanations for the incongruence of this finding with results from previous experiments are considered. • Incubation under an atmosphere of 100% CO 2 prevented the growth of the endophytic fungi present, despite drying of the wood. Fungal growth at low temperatures and at low oxygen tensions was less than could be explained through direct effects, and there was evidence for an indirect effect involving active host defence. Elevated temperatures favoured establishment of the early-colonizing ascomycete Biscogniauxia nummularia and the late-colonizing basidiomycete Coniophora puteana , while low oxygen tensions favoured colonization by Trichoderma sp. • The ecological significance of these observations is discussed.

Sudden infant death syndrome requires genetic predisposition, some form of stress and marginal malnutrition.

Over the past 30 years or more, the problem of sudden, unexplained death in infants (SIDS) has made little headway. Many hypotheses have been offered but the basic cause remains elusive. The only successful prevention has been made by the supine sleeping posture. There is still, however, a hard core of unexplained incidents. There is evidence that certain stress factors are involved, and there is good evidence that the tragedy has a familial or genetic tendency. The third factor necessary for the event is inefficient oxidation in brain cells induced most commonly by marginal malnutrition in pregnancy or after birth. The absence of any one or more of these three factors decreases risk to the point of extinction. Anything that impedes healthy oxidation, or accelerates energy utilization through responding to stress, increases the risk greatly. Improving the biochemical mechanisms through appropriate nutrition is by far the best defense.

Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5' sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as -86 to +217. Three upstream regions within -427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from -106 to -53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of -86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species.

Introns and their positions affect the translational activity of mRNA in plant cells.

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.

Mendel-GFDb and Mendel-ESTS: databases of plant gene families and ESTs annotated with gene family numbers and gene family names.

There is no control over the information provided with sequences when they are deposited in the sequence databases. Consequently mistakes can seed the incorrect annotation of other sequences. Grouping genes into families and applying controlled annotation overcomes the problems of incorrect annotation associated with individual sequences. Two databases (http://www.mendel.ac.uk) were created to apply controlled annotation to plant genes and plant ESTs: Mendel-GFDb is a database of plant protein (gene) families based on gapped-BLAST analysis of all sequences in the SWISS-PROT family of databases. Sequences are aligned (ClustalW) and identical and similar residues shaded. The families are visually curated to ensure that one or more criteria, for example overall relatedness and/or domain similarity relate all sequences within a family. Sequence families are assigned a 'Gene Family Number' and a unified description is developed which best describes the family and its members. If authority exists the gene family is assigned a 'Gene Family Name'. This information is placed in Mendel-GFDb. Mendel-ESTS is primarily a database of plant ESTs, which have been compared to Mendel-GFDb, completely sequenced genomes and domain databases. This approach associated ESTs with individual sequences and the controlled annotation of gene families and protein domains; the information being placed in Mendel-ESTS. The controlled annotation applied to genes and ESTs provides a basis from which a plant transcription database can be developed.

Isolation and characterization of the wheat prolyl isomerase FK506-binding protein (FKBP) 73 promoter.

The wheat FK506-binding protein (FKBP) 73 is a member of the peptidyl prolyl cis-trans isomerase gene family, which catalyses the interconversion between the cis and trans forms of the peptide bond preceding proline residues in proteins. A 3.5 kb sequence 5' upstream of the ATG codon of the wheat FKBP73 was isolated from a wheat genomic library, and characterized by deletion analysis and transient expression in wheat embryos. The 1517 bp fragment is referred to as the full promoter due to the maximal activity of the fused luciferase reporter gene. Sequence analysis revealed the presence of three abscisic acid (ABA)-responsive elements (ABREs) proximal to coupling elements (CE1-like), a putative lectin box, two putative binding sites for the myb transcription factor and a 36 bp fragment which exhibits 100% identity to the pSau3A9 clone located in the centromeric region of wheat chromosomes. In a transient expression assay the promoter preserved the tissue specificity described in vivo, namely it is expressed only in germinating embryos and young shoots. The promoter was induced 1.9-fold by ABA, the minimal promoter was designated at -221 and the TATA box located at -137. The inducibility by ABA and the expression during germination may indicate that FKBP73 belongs to the group of genes induced by ABA upon germination.

Evaluation of the biochemical effects of administration of intravenous nutrients using erythrocyte ATP/ADP ratios.

Regardless of clinical diagnosis, many acutely and chronically sick patients benefit from intravenous vitamins and minerals, which are usually administered in multiple infusions before observing obvious benefit. We hypothesized this effect was due to improved cellular energy, and attempted to find laboratory evidence via this study. Two groups of patients, chosen at random, received a single infusion of vitamins and minerals in two different dose schedules. Controls received no treatment. Study subjects were patients who presented specifically for a nutritional therapeutic approach, and although all were treated with multiple infusions, a single infusion was selected at random for this study. Thirty patients received a single infusion of a lower dose nutritional formula, sometimes known as a Myer's Cocktail (MC), and 34 had a single infusion of a higher dose nutritional I/V (NIV). Immediately prior to and after the infusion, blood was drawn and an erythrocyte ATP/ADP ratio (EADR) was determined. The results showed that in both infusion groups if the EADR was initially low, it would increase. If it were initially high, it would decrease. This effect was not observed in control subjects. Pre-test EADR boxplot analyses, derived from the results of each protocol, showed these results were statistically predictable. An analysis of variation (ANOVA) calculation indicated the differences were significant. The family error rate used was 0.05. We conclude that this regression of the EADR to the mean, as a result of either of the two infusions and not seen in control subjects, is biochemically significant.

Characterization of a gene encoding a single-subunit bacteriophage-type RNA polymerase from maize which is alternatively spliced.

Single-subunit RNA polymerases belonging to the T3/T7 bacteriophage family are thought to be common throughout eukaryotes. We report the isolation and characterization of a nucleus-encoded single-subunit RNA polymerase gene from maize. This gene is highly homologous to other single-subunit RNA polymerase genes from Arabidopsis, Chenopodium. yeast and Neurospora crassa involved in organellar transcription. Genomic Southern analysis reveals 10 to 15 hybridising fragments, suggesting that maize contains a small gene family. The isolated gene contains 19 exons and its genomic structure is highly conserved when compared to the three Arabidopsis homologues. Unlike the case in Arabidopsis, intron-12 of the maize bacteriophage-type RNA polymerase gene is alternatively spliced. Quantitative RT-PCR revealed that the resultant alternatively spliced transcript represents approximately 21 to 26% of the total polymerase mRNA in maize coleoptiles. The orthologous wheat bacteriophage-type RNA polymerase is also alternatively spliced and the intron exhibits 78% identity to maize intron-12. The conservation in alternative splicing between wheat and maize and its absence from Arabidopsis suggest a functional requirement for the alternatively spliced product.

The effect of altered codon usage on luciferase activity in tobacco, maize and wheat.

A comparison of the wild-type firefly luciferase reporter gene to a codon-modified gene, available from Promega, demonstrates that in tobacco cell cultures, an increase in G+C content of 1.8%, as a consequence of 36 A/T→G/C synonymous codon alterations and removal of the lysosomal targeting sequence, has no significant effect on expression. In maize Black Mexican Sweet cells and wheat scutellum, increases in activity of 14- to 23-fold and 53- to 59-fold, respectively, are obtained using the codon-modified luciferase with the UBI1 promoter and its leader intron. The observed increase in luc+ expression is most likely a consequence of differences in codon usage reflecting tRNA abundance rather than an increase in the efficiency of intron splicing resulting from the small increase in the G+C content of the coding sequence. This difference in light emission between the wild-type and codon-modified luciferases can be clearly visualised in a low-light imaging camera, making the latter a much more sensitive and useful reporter gene for detecting luciferase activity in vivo.

Strip-cankering of beech (Fagus sylvatica): Pathology and distribution of symptomatic trees.

The pathology and distribution of European beech trees bearing elongated bark lesions (strip-cankers) were investigated. Two types of canker were recognized: those on small trees (<40 cm diameter at breast height (dbh): 1·4 m above ground level) which bore fruit bodies of the xylariaceous ascomycete Biscogniauxia nummularia (Bull.) O. Kuntze, and those on larger specimens (>40 cm dbh) which were consistently associated with the diatrypaceous ascomycete Eutypa spinosa (Pers.) Tul. & C. Tul. All cankers were strictly annual, having formed during single growing seasons following periods of low water availability. The regional and local distribution of trees bearing lesions also appeared to be correlated with environmental conditions, being most severe where low rainfall or high temperatures had occurred. Population studies of the associated fungi, generally considered as saprotrophs, indicated the presence of unique genotypes within individual cankered trees and provided no evidence for the existence of pathotypes within either species. Within the decay columns which underlay canker surfaces, both B. nummularia and E. spinosa formed longitudinally extensive genets, implying non-mycelial spread in colonization. Suppression of both inter- and intraspecific incompatibility between fungi occurred in regions of canker decay columns with elevated water contents. The possible significance of coexistence between the ascomycetes B. nummularia and 'Hypoxylon purpureum' (sensu Sharland & Rayner, 1989b) and of the formation of heterokaryons by E. spinosa is discussed.

An analysis of the relative activities of a number of promoter constructs from genes which are expressed during late pollen development as determined by particle bombardment.

The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to theß-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S-ß-glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5'-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.