Search for unstable DNA in schizophrenia families with evidence for genetic anticipation.

Evidence for genetic anticipation has recently become an important subject of research in clinical psychiatric genetics. Renewed interest in anticipation was evoked by molecular genetic findings of a novel type of mutation termed "unstable DNA." The unstable DNA model can be construed as the "best fit" for schizophrenia twin and family epidemiological data. We have performed a large-scale Southern blot hybridization, asymmetrical PCR-based, and repeat expansion-detection screening for (CAG)n/(CTG)n and (CCG)n/(CGG)n expansions in eastern Canadian schizophrenia multiplex families demonstrating genetic anticipation. There were no differences in (CAG)n/(CTG)n and (CCG)n/(CGG)n pattern distribution either between affected and unaffected individuals or across generations. Our findings do not support the hypothesis that large (CAG)n/(CTG)n or (CCG)n/(CGG)n expansions are the major etiologic factor in schizophrenia. A separate set of experiments directed to the analysis of small (30-130 trinucleotides), Huntington disease-type expansions in individual genes is required in order to fully exclude the presence of (CAG)n/(CTG)n- or (CCG)n/(CGG)n-type unstable mutation.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.     

Uterine prostaglandin concentrations following fetal death in sheep

Concentrations of prostaglandin E (PGE), PGF and 6-oxo-PGF_1α (the hydrolytic product of PGI₂) were measured by radioimmunoassay (RIA) in myometrium, endometrium, cotyledons, amnion and chorioallantois taken from different uterine areas from chronically catheterized sheep bearing fetuses which had died 12–26 h previously (n=4) or 34–72 h previously (n=4). These two groups of animals were designated fetuses dead <30 h and >30 h respectively. The time of fetal death was assessed on the basis of fetal heart rate and blood gases. At the time of the tissue collection the ewes were between 123 and 130 days after mating. For comparative purposes, tissues also were collected from four sheep bearing live chronically catheterized fetuses at 130 days of gestation.For myometrium, concentration of PGF, PGE and 6-oxo-PGF_1α were significantly higher in sheep bearing dead fetuses, compared to those bearing live fetuses. Analysis of variance also showed a significant effect of uterine area on myometrial PGE concentrations, concentrations being higher in tubal areas than elsewhere. Concentrations of PGE, PGF and 6-oxo-PGF_1α were higher in endometrium taken from uteri containing dead fetuses. In cotyledons, concentrations of PGF and 6-oxo-PGF_1α but not PGE, were significant elevated following fetal death. Concentrations of 6-oxo-PGF_1α, but not PGE or PGF, were elevated in both chorioallantois and amnion of sheep bearing dead fetuses, compared to those bearing live fetuses. In association with elevated PG concentrations, there was a progressive increase in the frequency and maximum amplitude of uterine contractions. These results show that PG concetrations are elevated following fetal death in sheep, and suggest an association between elevated PG concentrations and delivery of the dead fetus.     

Multiple stressors: using the honeybee model BEEHAVE to explore how spatial and temporal forage stress affects colony resilience

The causes underlying the increased mortality of honeybee Apis mellifera colonies observed over the past decade remain unclear. Since so far the evidence for monocausal explanations is equivocal, involvement of multiple stressors is generally assumed. We here focus on various aspects of forage availability, which have received less attention than other stressors because it is virtually impossible to explore them empirically. We applied the colony model BEEHAVE, which links within‐hive dynamics and foraging, to stylized landscape settings to explore how foraging distance, forage supply, and “forage gaps”, i.e. periods in which honeybees cannot find any nectar and pollen, affect colony resilience and the mechanisms behind. We found that colony extinction was mainly driven by foraging distance, but the timing of forage gaps had strongest effects on time to extinction. Sensitivity to forage gaps of 15 days was highest in June or July even if otherwise forage availability was sufficient to survive. Forage availability affected colonies via cascading effects on queen's egg‐laying rate, reduction of new‐emerging brood stages developing into adult workers, pollen debt, lack of workforce for nursing, and reduced foraging activity. Forage gaps in July led to reduction in egg‐laying and increased mortality of brood stages at a time when the queen's seasonal egg‐laying rate is at its maximum, leading to colony failure over time. Our results demonstrate that badly timed forage gaps interacting with poor overall forage supply reduce honeybee colony resilience. Existing regulation mechanisms which in principle enable colonies to cope with varying forage supply in a given landscape and year, such as a reduction in egg‐laying, have only a certain capacity. Our results are hypothetical, as they are obtained from simplified landscape settings, but they are consistent with existing empirical knowledge. They offer ample opportunities for testing the predicted effects of forage stress in controlled experiments.     

Enzymatic synthesis of cytidine diphosphate diglyceride

Evidence is presented for the enzymatic formation of cytidine diphosphate diglyceride in microsomal preparations from guinea pig liver according to the reaction: CTP + phosphatidic acid right harpoon over left harpoon CDP-diglyceride + p-O-P. Conditions have been found in which the incorporation of labeled CTP into CDP-diglyceride is almost entirely dependent upon added phosphatidic acid. The incorporation of CMP into lipid is very slight. A substantial net synthesis of CDP-diglyceride takes place under these conditions. Some properties of the enzyme system are described.     

Toward using δ13C of ecosystem respiration to monitor canopy physiology in complex terrain

In 2005 and 2006, air samples were collected at the base of a Douglas-fir watershed to monitor seasonal changes in the delta13CO2 of ecosystem respiration (delta13C(ER)). The goals of this study were to determine whether variations in delta13C(ER) correlated with environmental variables and could be used to predict expected variations in canopy-average stomatal conductance (Gs). Changes in delta13C(ER) correlated weakly with changes in vapor pressure deficit (VPD) measured 0 and 3-7 days earlier and significantly with soil matric potential (psi(m)) (P value <0.02) measured on the same day. Midday G (s) was estimated using sapflow measurements (heat-dissipation method) at four plots located at different elevations within the watershed. Values of midday Gs from 0 and 3-7 days earlier were correlated with delta13C(ER), with the 5-day lag being significant (P value <0.05). To examine direct relationships between delta13C(ER) and recent Gs, we used models relating isotope discrimination to stomatal conductance and photosynthetic capacity at the leaf level to estimate values of stomatal conductance ("Gs-I") that would be expected if respired CO2 were derived entirely from recent photosynthate. We compared these values with estimates of Gs using direct measurement of transpiration at multiple locations in the watershed. Considering that the approach based on isotopes considers only the effect of photosynthetic discrimination on delta13C(ER), the magnitude and range in the two values were surprisingly similar. We conclude that: (1) delta13C(ER) is sensitive to variations in weather, and (2) delta13C(ER) potentially could be used to directly monitor average, basin-wide variations in Gs in complex terrain if further research improves understanding of how delta13C(ER) is influenced by post-assimilation fractionation processes.