New microsatellite markers for the abalone Haliotis midae developed by 454 pyrosequencing and in silico analyses.

Farming of Haliotis midae is the most lucrative aquaculture venture in South Africa. The genome of this species needs to be studied to assist in selective breeding programs aimed at increasing overall yield, and molecular markers will be required to attain this goal. We identified and characterized 82 polymorphic microsatellite loci by using repeat-enriched genomic libraries and high-throughput pyrosequencing technology. The observed number of alleles ranged from 2 to 21, expected heterozygosity from 0.063 to 0.968, observed heterozygosity from 0.000 to 1.000, and polymorphic information content from 0.059 to 0.934. Three loci gave significant hits to other haliotid genes and/or microsatellite loci; hits to genes were always located in the 5'/3'-UTR or intron region. Many of these newly designed markers would be useful for parentage, population and linkage studies.

Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.

BACKGROUND: Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling. RESULTS: To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. CONCLUSIONS: These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.

Insights into the structure of hepatocyte growth factor/scatter factor (HGF/SF) and implications for receptor activation.

The modular structure of HGF/SF offers a reductionist or 'divide and rule' approach to the analysis of structure and function. Domain deletion experiments have established that the N domain, kringle 1 and kringle 2 are essential for HGF/SF activity and that truncated variants containing the N domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) can exhibit partial agonistic or antagonistic activity depending on target cells. Comparative modelling has been used to predict the 3D structures of the six domains of HGF/SF. More recently, NMR methods have shown that the N domain has a novel fold, the charge distribution of which suggests a heparin binding site. Crystals of NK1 indicate the relationship of this domain to the kringle 1, offering further insights into the mechanism of domain interactions and receptor activation.

The effect of caffeine on cue exposure responses in ex-smokers.

The responses of ex-smokers to an experimental cue exposure trial, and the effect of caffeine on these responses, were compared with those of a matched group of control subjects in a placebo controlled single-blind cross-over design. In contrast to placebo, caffeine protected the ex-smokers from a surge of anxiety and rise in blood pressure associated with exposure to smoking-related cues. Caffeine had no significant effects on the control group at this dose (equivalent to a single cup of strong coffee). The results are discussed with reference to Stewart's conditioned appetitive motivational model of addiction. It is suggested that further work may identify caffeine as an adjunct to smoking relapse prevention measures.

Conversion pseudodementia in older people: a descriptive case series.

METHODS: This paper reports a case series of ten patients collected over a 12-year period of clinical work in old age psychiatry in the UK by the author. RESULTS: The core features of the syndrome are: apparent cognitive impairment, regression and increasing physical dependency beginning in late middle or early old age, without evidence for an organic dementia from investigations or from taking into account the course of the illness. The syndrome is more common in women from a higher socio-economic background with past psychiatric histories dominated by depressive symptoms. The syndrome usually progresses to the point where long term institutional care is needed although the mean survival from onset is 13 years. CONCLUSIONS: The author suggests that conversion pseudodementia in older people is caused by a catastrophic reaction to cumulative loss in later life in individuals who have predisposing borderline and narcissistic personality traits. Treatment using psychotherapeutic approaches may limit the progression of the syndrome if it is recognised at an early stage.

One hundred cases of attempted suicide in the elderly.

BACKGROUND: Despite the high suicide rate in the elderly, there is a relative lack of information on the longer-term outcome of elderly people who have attempted suicide, particularly their psychiatric and physical morbidity and mortality. METHOD: Comprehensive demographic and psychiatric data were available on 100 consecutive referrals to a liaison psychiatric service of patients over 65 years of age who attempted suicide between 1989 and 1992. Detailed follow-up in 1994 included the interviewing of survivors using GMS-AGECAT. RESULTS: Of the 64 women and 36 men, with a mean age of 75.8 years, 42 subjects were dead at follow-up; 12 were suspected suicides, five having died as a delayed result of their index attempt. Twelve women made a further non-lethal suicide attempt. All five male repeat attempts proved fatal. CONCLUSIONS: Elderly people who attempt suicide have a high mortality both from completed suicide and death from other causes. The completed suicide rate is at least 1.5% per year, and the repetition rate is 5.4% per year. Those at risk of further self-harm are likely to be in contact with psychiatric services and to be suffering from persistent depression.

A survey of private nursing home residents in Weston Super Mare.

A representative sample of 351 patients in Private Nursing Homes in Weston Super Mare was surveyed with regard to their origins, admission assessment and continuing care, with particular reference to the nature of medical review during their residency. The results suggest inadequate medical care especially for 'imported' patients and those admitted for psychiatric reasons.

Domain structure of hepatocyte growth factor/scatter factor (HGF/SF).

The modular structure of hepatocyte growth factor/scatter factor (HGF/SF) has facilitated structure-function analysis. Domain deletion experiments have established that the N-domain, kringle 1 and kringle 2 are essential for HGF/SF activity on target cells and that, conversely, truncated variants containing the N-domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) exhibit partial agonistic or antagonistic activity depending on target cells and the presence of full length HGF/SF. The 3D structures of the six domains of HGF/SF have been modelled on the structure of homologues, offering interesting insights into putative mechanisms of domain interactions, receptor binding and activation. The predictions offered by such models are currently assessed by protein engineering techniques and will ultimately be measured against experimental structures.

Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding.

Although ligand-induced receptor dimerization is a common prerequisite for receptor activation, the mode by which different growth factors bind their receptors and cause them to dimerize varies considerably. Here we report the crystal structure at 2.5 A resolution of NK1, a receptor-binding fragment and a natural splice variant of hepatocyte growth factor/scatter factor (HGF/SF). NK1 assembles as a homodimer in the asymmetric unit, revealing a novel mode of growth factor dimerization produced by close packing of the N domain of one subunit and the kringle domain of the other, thus bringing the two linkers in close proximity. The structure suggests the presence of a binding site for heparan sulfate chains and a mechanism by which the NK1 dimer may engage two receptor molecules through clusters of amino acids located on each protomer and on opposite surfaces of the homodimer. We also report that short (14-mer) heparin fragments effectively dimerize NK1 in solution, implying that heparan sulfate chains may stabilize the NK1 dimer. These results provide a basis for the agonistic activity of NK1 and have implications for the mechanism of receptor binding of HGF/SF.