A computational approach to identify genes for functional RNAs in genomic sequences.

Currently there is no successful computational approach for identification of genes encoding novel functional RNAs (fRNAs) in genomic sequences. We have developed a machine learning approach using neural networks and support vector machines to extract common features among known RNAs for prediction of new RNA genes in the unannotated regions of prokaryotic and archaeal genomes. The Escherichia coli genome was used for development, but we have applied this method to several other bacterial and archaeal genomes. Networks based on nucleotide composition were 80-90% accurate in jackknife testing experiments for bacteria and 90-99% for hyperthermophilic archaea. We also achieved a significant improvement in accuracy by combining these predictions with those obtained using a second set of parameters consisting of known RNA sequence motifs and the calculated free energy of folding. Several known fRNAs not included in the training datasets were identified as well as several hundred predicted novel RNAs. These studies indicate that there are many unidentified RNAs in simple genomes that can be predicted computationally as a precursor to experimental study. Public access to our RNA gene predictions and an interface for user predictions is available via the web.

Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation.

Huntington's disease (HD) is an autosomal dominant progressive and fatal neurodegenerative brain disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the gene. Presymptomatic Huntington's disease patients often exhibit cognitive deficits before the onset of classical symptoms. To investigate the possibility that changes in synaptic plasticity might underlie cognitive impairment in HD, we examined hippocampal synaptic plasticity and spatial cognition in a transgenic mouse (R6/2 line) expressing exon 1 of the human Huntington's disease gene containing an expanded CAG repeat. This mouse exhibits a progressive and fatal neurological phenotype that resembles Huntington's disease. We report that R6/2 mice show marked alterations in synaptic plasticity at both CA1 and dentate granule cell synapses, and impaired spatial cognitive performance in the Morris water maze. The changes in hippocampal plasticity were age dependent, appearing at CA1 synapses several weeks before they were observed in the dentate gyrus. Deficits in synaptic plasticity at CA1 synapses occurred before an overt phenotype. This suggests that altered synaptic plasticity contributes to the pre-symptomatic changes in cognition reported in human carriers of the Huntington' disease gene. The temporal and regional changes in synaptic plasticity within the hippocampus mirror the appearance of neuronal intranuclear inclusions, suggesting a relationship between polyglutamine aggregation and dysfunction.

Sensitive new in vitro bioassay for melanocyte-stimulating activity using the skin of Anolis carolinensis.

An accurate, highly reproducible and sensitive bioassay for melanocyte-stimulating hormone (MSH) using the skin of Anolis carolinensis in vitro is described. The time taken for green Anolis skin fragments to change to a specific, visually assessed, green-brown color is dose-related, and this forms the basis of the new assay. The method is simple to perform, and 1 person may assay 20 samples in a day using the dorsal skin from a single adult lizard. The mean dose-response ranges between 48 X 10(-12) and 375 X 12(-12) M (38 to 625 pg/ml). Using the assay, alpha-MSH, beta-MSH, ACTH (4-10), and ACTH (1-10) were equipotent on a molar basis. For repeated bioassay of rat pituitary extracts, the dose-response curves were highly significant, and only 1 of the 9 pituitary dose-response curves deviated significantly from the slope of the standard alpha-MSH curve. The index of precision, lambda, for the 9 pituitary bioassays ranged between 0.037 and 0.081, while the mean 95% fiducial limits were -6.6 and 7.1% on either side of the estimated potency. The new rate method is compared with an earlier quantal method which also uses the isolated skin of Anolis carolinensis. The quantal method does not have dose-response characteristics and is therefore less accurate and reproducible than the new method; the coefficient of variation for repeated bioassay of the same pituitary extracts ranged from between 12 to 20% for the quantal method and between 2.9 to 5.7% for the new rate method.

A comparison of methods for obtaining high yields of pure immunoglobulin from severely haemolysed plasma.

Ammonium sulphate, DEAE ion exchange chromatography, and polyethylene glycol (PEG) were compared for their ability to isolate and purify large quantities of immunoglobulins from severely haemolysed plasma. PEG, which produced the highest yield and purity, proved to be the only method which completely removed the haemoglobin.

The association between the melanocyte-stimulating hormone receptor and the alpha 2-adrenoceptor on the Anolis melanophore.

1 The primary effect of catecholamines was to lighten Anolis skin previously darkened by alpha-melanocyte-stimulating hormone (alpha-MSH). In concentrations above 10(-7) M noradrenaline, 10(-6) M adrenaline and 10(-5) dopamine, darkening of subpopulations of melanophores occurred. Subsequent experiments were concerned with the effect of low catecholamine concentrations on alpha-MSH action. 2 The relationship between MSH receptors and alpha-adrenoceptors on the Anolis melanophore was studied by a kinetic approach using the rate bioassay method and by use of alpha-adrenoceptor agonists and antagonists. 3 alpha-MSH dose-response curves were shifted, in parallel, to the right in the presence of the catecholamines, noradrenaline, adrenaline and dopamine, and Lineweaver-Burke plots and Arunlakshana-Schild plots indicated that the catecholamines antagonized MSH action by a competitive mechanism. 4 Phentolamine had an inhibitory effect on the action of adrenaline but not on the action of MSH. Therefore MSH and catecholamine actions were mediated by separate receptors. 5 The classical kinetics of competition are not confined to competition at a single receptor. 6 The alpha-adrenoceptor was defined as the alpha 2-subtype since (a) the alpha 2-selective agonist, clonidine, was found to mimic catecholamine action. (b) The alpha 2-selective antagonist, yohimbine, blocked the actions of clonidine and adrenaline. (c) The alpha 1-selective antagonist, prazosin, had negligible blocking effects on adrenaline and clonidine. 7 We conclude that a close association exists between the separate MSH receptor and alpha 2-adrenoceptor on the Anolis melanophore. The competition that takes place between MSH and catecholamines must occur after hormone-receptor interaction, possibly through a common adenylate cyclase moiety oppositely controlled by the two receptors involved.

Morquio syndrome (MPS IVA) and hypophosphatasia in a Hutterite kindred.

A patient is described who has Morquio syndrome (MPS IVA). He is a member of the Hutterite Brethren and genealogic analysis discloses a high inbreeding coefficient for the proband. The proband's sibship is segregating two autosomal recessive disorders, ie, MPS IVA and infantile hypophosphatasia. Two other families each have one or the other of these diseases but not both. The three families are distantly related.

Effects of medium-chain triglyceride feeding on energy balance in adult humans.

In recent years, the metabolism of triglycerides has attracted much attention. Oxidation of fatty acids is an essential energy supply, especially when glucose supply is limited. In the present study, the effect of a 3-day high medium-chain triglyceride (MCT; 51% of calories), low carbohydrate intake on plasma glucose and amino acid, and urinary organic acid levels, including dicarboxylic and tricarboxylic acid cycle intermediates, was determined in eight normal adult volunteer subjects. Urine was collected at baseline and at 48 to 72 hours for amino acid and organic acid levels, and plasma collected at 0 and 72 hours for glucose and amino acid concentration. The MCT diet increased urinary levels of dicarboxylic acids (adipic 8-, suberic 65-, sebacic 284-fold) and keto acids (acetoacetate and beta-hydroxybutyrate, 67.5-fold); alanine and lactate were decreased 2.5- and 4-fold, respectively, while pyruvate, other amino acids and citric acid intermediates remained unchanged. Plasma amino acid levels were unchanged, while the plasma glucose levels decreased by 8% from baseline. The loss of calories as urinary dicarboxylic acids and keto acids, although increased during the MCT diet, was less than 1% of the daily caloric intake. The data suggest MCT sustain energy expenditure through medium-chain fatty acid (MCFA) oxidation with no decrease in citric acid cycle intermediates, while sparing protein oxidation.

Des-acetyl MSH and gamma-MSH act as partial agonists to alpha-MSH on the Anolis melanophore.

The biological activities of alpha-MSH des-acetyl MSH, gamma-MSH and LPH37-58 were compared using the Anolis rate method of bioassay. Dose-response data showed LPH37-58 to be equipotent with alpha-MSH, but des-acetyl MSH and gamma-MSH were found to be much less active. The effect of LPH37-58 was additive to that of alpha-MSH, indicating that LPH37-58 is a full agonist of alpha-MSH. The lower potency peptides des-acetyl MSH and gamma-MSH reduced the effect of alpha-MSH and are, therefore, partial agonists of alpha-MSH. The action of MSH peptides in vivo may be modulated by interaction with agonists.

Alpha-MSH and coat color changes in the mouse.

The effect of alpha-MSH on coat color was examined in viable yellow mice (C3H/He-A*vy). These mice normally grow a coat of darkly pigmented hair at puberty. This darkening effect was also evident in hair that grew in a region that had been plucked at 13 days of age. Administration of alpha-MSH increased the darkness of this hair and the hair which grew naturally in an unplucked area. However, the natural coat darkening that occurred at puberty was not associated with an increase in plasma immunoreactive alpha-MSH levels. Moreover, although bromocryptine, a dopamine agonist that inhibits alpha-MSH release from the pituitary reduced the darkness of the coat that grew after plucking the reduction in coat darkening was unrelated to changes in plasma alpha-MSH. Nevertheless, this effect of bromocryptine was reversed when alpha-MSH was administered together with the drug. Apomorphine had no effect on coat darkening and produced only a slight decrease in plasma alpha-MSH. Melatonin reduced coat darkening slightly but, like apomorphine, had little effect on plasma alpha-MSH concentrations. Although alpha-MSH may have a physiological role in coat darkening in the C3H/He-A*vy mouse at puberty the response seems to be unrelated to an increase in circulating alpha-MSH. Thus, other factors, such as changes in melanocyte sensitivity to alpha-MSH or inhibitory mechanisms that prevent coat darkening during prepubertal and adult life may be involved in regulation of coat color in the viable yellow mouse.

Melanotrophin-potentiating factor (MPF) potentiates MSH-induced melanogenesis in hair follicle melanocytes.

Melanotrophin-potentiating factor (MPF) is a fragment of human beta-lipotrophin (LPH 88-91) which potentiates the action of alpha-MSH on Anolis skin. In the present study, we investigated the effect of MPF on MSH-induced melanogenesis. Pooled hair follicle scrapings from Siberian hamsters (Phodopus sungorus) were incubated for 48 hours with or without alpha-MSH and/or MPF. Melanogenesis was monitored by measuring tyrosinase activity and melanin accumulation. 10-8 M MPF potentiated the effect of no effect on melanogenesis, but 10-9 to 10-7 M alpha-MSH caused a dose-related increase. 10-8 M MPF potentiated the effect of each dose of alpha-MSH. Thus MPF potentiated MSH action on mammalian melanogenesis as well as on reptilian melanosome dispersion. Although each of these processes involve different intracellular responses the receptor mechanisms involved in each may therefore be the same.

Interleukin-2-dependent T lymphocytes for the diagnosis and investigation of inherited metabolic disorders.

Interleukin-2-dependent T cell lines can be started from as little as 1 ml of peripheral blood and expanded to 50 x 10(6) cells within 12 days. These cells represent an easily obtainable source of nucleated tissue with certain advantages over skin fibroblasts or transformed B lymphoblastoid cells for the rapid diagnosis and study of inherited metabolic disorders. A panel of 19 enzymes were assayed in IL-2-dependent T lymphocytes and the diagnosis of four enzyme deficiencies is demonstrated using T lymphocytes.

Melanocyte-stimulating hormone--mimetic action of the phenothiazines.

We have compared the melanophore-stimulating action of four phenothiazines, trifluoperazine, perphenazine, chlorpromazine, and prochlorperazine, with alpha-MSH on the skin of the lizard Anolis carolinensis, using a new rate method of bioassay. The dose-response curves for the phenothiazines were parallel to that of alpha-MSH, and when given together alpha-MSH and chlorpromazine were additive. The phenothiazines may therefore stimulate melanosome dispersion in the lizard skin by the same mechanism as alpha-MSH; a MSH-mimetic action of phenothiazines may similarly explain their pigmentary action in man. The pigmentary potency of the phenothiazines corresponded with their therapeutic potency in man; this is in keeping with a neuro-regulatory role for MSH peptides and suggests a possible therapeutic use for them.

The use of an oxygen bomb as an alternative method for digestion of fish tissue for total mercury analysis.

One of the major time consuming steps in many of the methods used for the analysis of fish tissue for total mercury is the wet oxidation by concentrated acids. This paper compares the use of an oxygen bomb with the nitric/sulfuric acid wet oxidation technique for the determination of total mercury in fish tissue. No significant differences existed in the concentrations obtained by wet oxidation and bomb oxidation with absorption by deionised distilled water. When only a small number of samples (< 5) is to be analysed the oxidation bomb is quicker than published techniques. The oxygen bomb technique lacks the hazardous problems associated with the use of concentrated acids and produces a digest suitable for the determination of other elements but has a requirement for added capital cost.

Physiological amino acid data management: quantitation, assessment, reporting and storage.

A series of routines, written in BASIC, have been developed to aid in the analysis, reporting and storage of physiological amino acid data used in the diagnosis and management of inherited metabolic disorders. The concentrations of 44 compounds are determined for three types of physiological samples: plasma, urine or cerebral spinal fluid. The programs facilitate the editing of numerical data, the creation of a patient and sample information file to be merged with the results, the flagging of abnormal results, the addition of diagnostic or interpretive comments and the generation of hard copy reports. Files containing the foregoing information provide records which may be manipulated using data base programs for further analysis.

Assessing capacity for diagnosing tuberculosis in children in sub-Saharan African HIV care settings.

Research on the prevalence of pediatric-specific tuberculosis (TB) diagnostics in sub-Saharan Africa is scarce. We assessed the availability of pediatric TB diagnostic tests at 651 pediatric human immunodeficiency virus care and treatment sites across nine African countries: 54% of the sites had access to sputum culture capacity and 51% to chest X-ray services. While 87% of sites had access to smear microscopy, only 6% had the capacity to perform sputum induction and 5% to perform gastric aspirate. These findings confirm that diagnostic resources for the accurate diagnosis of pediatric TB are limited. Capacity-building initiatives to improve sputum collection in children are urgently required.

The role of mast cells in the structural alterations of the airways as a potential mechanism in the pathogenesis of severe asthma.

Mast cells, traditionally regarded as effector cells of the immune system, have more recently been demonstrated to be key figures in initiating, developing and sustaining complex pathophysiological processes underlying asthma and other allergic diseases. Asthma is characterised by airway inflammation alongside a disturbance to airway physiology manifesting as variable airflow obstruction and airway hyper-responsiveness (AHR). Evidence has emerged that mast cells influence airway function by forming close intercellular relationships with different structural components of the airway wall. In asthma, mast cells are seen to localise to the airway epithelium, to mucous glands and to the airway smooth muscle (ASM). It is mast cell-ASM interaction that is most fundamental to the asthma phenotype and many mast cell mediators have been demonstrated to have important effects on ASM function. In asthma, alongside the inflammatory and physiological changes, structural changes occur to the airway wall in the form of denudation of the epithelium, goblet cell and mucous gland hyperplasia, subepithelial fibrosis, abnormal extracellular matrix (ECM) deposition, vascular proliferation and increased ASM mass. There are many ways in which mast cells can contribute to these structural changes through direct cell to cell communication and more indirectly through mediator release. Mast cells exhibit an array of diverse functions and roles and are fundamental to our current understanding of asthma pathogenesis including severe asthma. Novel targeting of mast cells and their mediators therefore should offer significant therapeutic potential in the treatment of asthma.

Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase.

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.

Environmental stimulation increases survival in mice transgenic for exon 1 of the Huntington's disease gene.

Mice transgenic for the first exon of the human Huntington's disease (HD) gene carrying an expanded CAG repeat expansion (R6/2 line) develop a progressive neurologic phenotype with symptoms resembling those seen in HD. The overt symptoms of R6/2 mice worsen with age, resulting in a rapid decline in health and premature death between 13 and 18 weeks of age. In this study, we characterized the onset and progression of the overt phenotype in R6/2 mice and examined factors that affect the phenotype and life expectancy of these mice. In particular, the effects of altering home cage environment, through changing feeding regimes and providing environmental stimulation, were investigated. We show that changes in feeding regimes significantly improved the general well-being and life expectancy of R6/2 mice. Furthermore, we find that various forms of environmental stimulation, including regular behavioral testing, significantly improved the survival of R6/2 mice over and above that resulting from the enhanced feeding regime. The fact that environmental stimulation improves the health and life expectancy in R6/2 mice not only enables the mice to serve as more useful research tools, but also suggests that environmental stimulation may have a beneficial impact on the progression of HD in patients.

Dopamine modulates the susceptibility of striatal neurons to 3-nitropropionic acid in the rat model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by chorea, psychiatric disturbances, and dementia. The striatum is the primary site of neuronal loss in HD; however, neither the mechanism of neurodegeneration nor the underlying cause of the selectivity for the striatum is understood. Chronic systemic injection of 3-nitropropionic acid (3-NP) into rats induces bilateral striatal lesions with many neuropathological features of HD and is widely used as a model of HD. In this study we examine the role striatal dopamine plays in 3-NP-induced striatal toxicity. The effect of elevated striatal dopamine levels on 3-NP toxicity was examined by using acute administration of methamphetamine. After 7 d of 3-NP treatment, a single low dose of methamphetamine markedly increased the frequency of striatal lesion formation. This effect was mediated via dopamine receptors because it could be blocked by the administration of dopamine receptor antagonists. The effect of decreased striatal dopamine on 3-NP toxicity was examined by lesioning the nigrostriatal dopamine input to one striatum 7 d before 3-NP treatment was started. Removal of the dopamine input protected the denervated striatum from the neurotoxic effects of systemic 3-NP but did not prevent the formation of lesions in the intact striatum. Thus the formation of 3-NP lesions is critically dependent on an intact dopamine input. Our data show that dopamine plays an important role in the formation of 3-NP lesions. We suggest that modulation of the dopaminergic system should be reevaluated as a potential drug target in the treatment for HD.