The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Frequency of cytokine polymorphisms in populations from western Europe,Africa, Asia,the Middle East and South America

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.     

Vagal-enteric interface: vagal activation-induced expression of c-Fos and p-CREB in neurons of the upper gastrointestinal tract and pancreas

Many gastrointestinal and pancreatic functions are under strong modulatory control by the brain via the vagus nerve. To start identifying location and neurochemical phenotype of the enteric neurons receiving functional vagal efferent input, we activated vagal preganglionic neurons either by electrical or chemical stimulation and examined the expression of phosphorylated CREB (c-AMP response element binding protein) and the immediate early gene c-Fos. There was no spontaneous expression of both markers in the pancreas and considerable spontaneous expression of p-CREB but not Fos in the upper GI-tract. Unilateral electrical vagal stimulation-induced p-CREB was found in 40% of neurons in the head of the pancreas. Fos expression was found in 70-90% of neurons in the esophagus and stomach, in 20-30% of myenteric plexus neurons and 5-15% in submucosal neurons of the proximal duodenum. Double-labeling experiments showed that a majority of pancreatic neurons and about 25-35% of neurons in the stomach and duodenum contain NADPH-diaphorase and that many of these receive functional vagal input. Other neurons that can be vagally activated contain gastrin-releasing peptide or calretinin. Chemical stimulation of the dorsal surface of the caudal brainstem with the stable TRH analog RX77368 resulted in selective activation of vagal efferents with expression of Fos in a small number of gastric myenteric plexus neurons. The results demonstrate the suitability of this method to investigate magnitude and local distribution of vagal input to the enteric nervous system as well as specificity of its neurochemically coded pathways. They represent the first step in the identification of function-specific units of parasympathetic vagal outflow.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Neonatal impact of leukemia inhibitory factor on neurobehavioral development in rats

Cytokines have been implicated in the etiology or pathology of various psychiatric diseases of developmental origin such as autism and schizophrenia. Leukemia inhibitory factor (LIF) is induced by a variety of brain insults and known to have many influences on mature and immature nervous system. Here, we assessed the neurobehavioral and pathological consequences of peripheral administration of LIF in newborn rats. Subcutaneous LIF injection induced STAT3 phosphorylation in many brain regions and increased glial fibrillary acidic protein (GFAP) immunoreactivity in the neocortex, suggesting that LIF had direct effects in the central nervous system. The LIF-treated rats displayed decreased motor activity during juvenile stages, and developed abnormal prepulse inhibition in the acoustic startle test during and after adolescence. They displayed normal learning ability in active avoidance test, however. Brain neuronal structures and startle responses were grossly normal, except for the cortical astrogliosis during neonatal LIF administration. These results indicate that LIF induction in the periphery of the infant has a significant, but discrete impact on neurobehavioral development.     

The pattern of function-related regional cerebral blood flow investigated by single photon emission tomography with 99mTc-HMPAO in patients with presenile Alzheimer's disease and Korsakoff's psychosis

Single photon emission tomography (SPET) with the lipophilic blood flow marker 99mTc-hexamethyl propyleneamine oxime (99mTc-HMPAO) has been used to determine regional uptake of radiolabel into brain regions of patients with presenile Alzheimer's disease and Korsakoff's psychosis, and age-matched controls. Using occipital cortical uptake as reference area, the pattern of relative regional cerebral blood flow (rCBF) was determined in other cortical areas and basal ganglia. In Alzheimer's disease, reduction in rCBF occurred most strikingly in posterior temporal and parietal areas. By contrast, in Korsakoff's psychosis, posterior temporal rCBF was maintained, although there was a trend to reduced tracer uptake in other cortical areas. These impairments of flow were correlated with impairments of neuropsychological function. In Alzheimer's disease, left posterior temporal and left parietal regions in particular showed rCBF to be strongly correlated with most aspects of cognitive function. In Korsakoff's psychosis, however, impaired flow in frontal regions was correlated with impaired performance on tests of memory and orientation. The findings in Alzheimer's disease show quantitative parallels with those from studies using Positron Emission Tomography (PET), and extend our understanding of the relationship between cognition and regional brain function in dementia. The findings in Korsakoff's psychosis offer the first direct evidence linking frontal lobe dysfunction with the cognitive impairment seen in the disorder.     

Stimulatory and suppressive effects of infection of dendritic cells with HIV-1.

Two effects of HIV infection on human dendritic cells (DC) in vitro have been examined. The first was the stimulation of primary responses to HIV antigens in autologous lymphocytes from normal donors. When DC were exposed to HIV (10(4) TCID/10(5) cells) for up to 24 h before addition to autologous lymphocytes, a marked primary proliferative response to the virus was observed. No proliferative response was seen when the period of pre-exposure of DC to virus was extended. Cytotoxic T cells specific for HIV-infected target cells developed in stimulated cultures. The second effect of HIV infection of DC was to block responses to other antigens, such as alloantigens and the recall antigens tetanus toxoid and influenza virus. This inhibitory effect was only evident when the DC were exposed to HIV for longer than 24 h before being added to cultures. These in vitro studies suggest that infection of DC can produce both stimulatory and inhibitory responses in lymphocytes. Such effects operating through DC might underlie in vivo activity of HIV both in stimulating the proliferation of lymphocytes (e.g., in persistent generalised lymphadenopathy) and in the development of immunosuppression.     

Parasite-susceptibility phenotypes of F1 Biomphalaria glabrataprogeny derived from interbreeding Schistosoma mansoni-resistant and -susceptible snails

In an effort to investigate the 'flow' of parasite-resistance genes through laboratory snail populations, we determined the susceptibility of progeny snails from freely interbreeding parasite-susceptible and parasite-resistant parents. Five parental populations of Biomphalaria glabrata were used to generate the progeny snails. Three of them contained different proportions of Schistosoma mansoni-susceptible albino snails (NMRI stock) and S. mansoni-resistant pigmented snails (BS-90), while single stock controls comprised the other two parental populations. F(1) snails from each parental population were exposed to S. mansoni miracidia. Some of the progeny snails were exposed as juveniles, others as adults. According to Hardy-Weinberg principle predictions, the F(1) generation from the three pigmented/albino parental populations displayed higher than expected numbers of pigmented (resistant) snails and lower than expected numbers of albino (susceptible) snails. Among the assumptions of the Hardy-Weinberg principle that were not met within these populations could include non-random mating, unequal fecundity, different hatching and survival rates of different genotypes, or other life-history differences between snail stocks. It is clear, though, that for these two laboratory snail stocks there is no fitness cost attached to genetic resistance to the parasite.     

Familial Similarities of Changes in Cognitive, Behavioral, and Physiological Variables in a Cardiovascular Health Promotion Program

A number of studies have demonstrated that physiological andbehavioral cardiovascular disease (CVD) risk factors aggregatewithin families. This fact, and the potential mediating rolethat the family plays in behavior change, have led to the developmentof family-based CVD risk reduction programs, including the SanDiego Family Health Project. The aggregation of behavioral,physiological, and cognitive changes within families was assessedduring a 1-year intervention. We found evidence of modest butsignificant aggregation of change. There was more aggregationof change in behavioral variables than in physiological or cognitivevariables. More significant correlations were found among 3-dayfood record measures than among 24-hour recall dietary measures,suggesting an influence of assessment method. Aggregation ofchange within families was stronger within generations thanacross generations. These data point to the importance of involvingall age groups in health promotion programs.