Identification of loci determining susceptibility to the lethal effects of amyloid precursor protein transgene overexpression

Phenotypes produced by expression of human amyloid precursor protein (APP) transgenes vary depending on the genetic background of the mouse. FVB/N mice overexpressing human APP695 develop a central nervous system disorder and die prematurely, precluding development of Abeta peptide amyloid plaques. 129S6 mice are resistant to the lethal effects of APP overexpression, allowing sufficient levels of Abeta expression for the development of amyloid plaques and age-dependent memory deficits. To identify the genes that determine susceptibility or resistance to APP we analyzed crosses involving FVB/NCr and 129S6.Tg2576 mice that overexpress 'Swedish' mutant (K670N, M671L) APP695. APP transgene-positive FVB129S6F1 (F1) mice are resistant to the lethal effects of APP overexpression, so FVBxF1 backcross and F2 intercross offspring were produced. Analysis of age of death as a quantitative trait revealed significant linkage to loci on proximal chromosome 14 and on chromosome 9; 129S6 alleles protect against the lethal effects of APP. Within the chromosome 14 interval are segments homologous to regions on human chromosome 10 that have been linked to late onset Alzheimer's disease or to levels of Abeta peptide in plasma. However, analysis of plasma Abeta peptide concentrations at 6 weeks in backcross offspring produced no significant linkage. Similarly, elevation of human Abeta peptide concentrations by expression of mutant presenilin transgenes did not increase the proportion of mice dying prematurely, suggesting that early death reflects effects of APP or fragments other than Abeta.     

Essential light chain of Drosophila nonmuscle myosin II

Summary We have cloned and sequenced a cDNA encoding the essential (alkaline) light chain of nonmuscle myosin from Drosophila melanogaster. The protein predicted from the cDNA matches partial amino acid sequence derived from essential light chain protein that copurifies with native nonmuscle myosin heavy chain. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. We identified the most conserved residues among essential light chain sequences from multiple phyla and present their locations on the crystallographic structure of striated muscle essential light chain. This highlights several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 and we discuss prospects for genetic analysis in this region.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Development of vaccination strategies that elicit broadly neutralizing antibodies against human immunodeficiency virus type 1 in both the mucosal and systemic immune compartments

The antigenic diversity, rapid genetic integration into host cell DNA, and immune evasion tactics of human immunodeficiency virus type 1 (HIV-1) create formidable obstacles to the development of an effective vaccine against it. In spite of this, the advent of conformationally constrained HIV-1 Env and gp120 immunogens has made it feasible to formulate HIV-1 vaccines that induce broadly cross-reactive neutralizing antibodies and afford protection through humoral mechanisms. This paper reviews recent advances made by the authors toward the development of an HIV-1 vaccine that elicits such antibodies in both the mucosal and systemic immune compartments.     

The effect of gravity on the horizontal and vertical vestibulo-ocular reflex in the rat

Horizontal and vertical eye movements were recorded in alert pigmented rats using chronically implanted scleral search coils or temporary glue-on coils to test the dependence of the vestibulo-ocular reflex (VOR) upon rotation axis and body orientation. The contributions of semicircular-canal versus otolith-organ signals to the VOR were investigated by providing canal-only (vertical axis) and canal plus otolith (horizontal axis) stimulation conditions. Rotations that stimulated canals only (upright yaw and nose-up roll) produced an accurate VOR during middle- and high-frequency rotations (0.2-2 Hz). However, at frequencies below 0.2 Hz, the canal-only rotations elicited a phase-advanced VOR. The addition of a changing gravity stimulus, and thus dynamic otolith stimulation, to the canal signal (nose-up yaw, on-side yaw, and upright roll) produced a VOR response with accurate phase down to the lowest frequency tested (0.02 Hz). In order to further test the dependence of the VOR on gravitational signals, we tested vertical VOR with the head in an inverted posture (inverted roll). The VOR in this condition was advanced in phase across all frequencies tested. At low frequencies, the VOR during inverted roll was anticompensatory, characterized by slow-phase eye movement in the same direction as head movement. The substantial differences between canalonly VOR and canal plus otolith VOR suggest an important role of otolith organs in rat VOR. Anticompensatory VOR during inverted roll suggests that part of the otolith contribution arises from static tilt signals that are inverted when the head is inverted.     

Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation

The aim of this study was to examine the kinetic profile of bioactive TNF levels in aqueous humour of rabbit eyes undergoing corneal allograft rejection and to investigate the effect of locally blocking TNF activity after corneal transplantation. In a rabbit corneal transplantation, endothelial allograft rejection was identified and correlated with increase in central graft thickness. Samples of aqueous humour obtained on alternate days following transplantation were tested for TNF mRNA and bioactive TNF protein. To investigate the effect of locally blocking TNF activity in allograft recipients, the fusion protein TNFR-Ig was administered by injections into the anterior chamber after transplantation. Pulsatile increases in levels of this cytokine were found in 14 of 15 allograft recipients. Peaks of TNF bioactivity preceded by varying intervals the observed onset of rejection in allograft recipients. TNF levels were not elevated in aqueous humour from corneal autograft recipient controls or in serum of allografted animals. mRNA levels were elevated before onset of and during clinically observed allograft rejection. In three of seven animals receiving TNFR-Ig injections on alternate days from day 8 to day 16 post-transplant, clear prolongation of corneal allograft survival was demonstrated. Bioactive TNF is present in aqueous humour following rabbit corneal allotransplantation. Rather than correlating directly with endothelial rejection onset, pulsatile peak levels of TNF precede and follow the observed onset of endothelial rejection. Blockade of TNF activity prolongs corneal allograft survival in some animals, indicating that this cytokine may be a suitable target in local therapy of corneal allograft rejection.     

Analysis of Connective Tissue Progenitor Cell Behavior on Polydimethylsiloxane Smooth and Channel Micro-Textures

Growth of human connective tissue progenitor cells (CTPs) was characterized on smooth and microtextured polydimethylsiloxane (PDMS) surfaces. Human bone marrow derived cells were cultured for nine days under conditions promoting osteoblastic differentiation on Smooth PDMS and PDMS Channel microtextures (11 m high, 45 m wide channels, and separated by 5 m wide ridges). Glass tissue culture dish surfaces were used as controls. Cell numbers per colony, cell density within colonies, alignment of cells, area of colonies, and colony shapes were determined as a function of substrate surface topography. An alkaline phosphatase stain was used as a marker for osteoblastic phenotype. CTPs attached, proliferated, and differentiated on all surfaces with cell process lengths of up to 80 m. Cells on the Smooth PDMS and control surfaces spread and proliferated as colonies in proximity to other cells and migrated in random directions creating colonies that covered significantly larger areas (0.96 and 1.05 mm², respectively) than colonies formed on PDMS Channel textures (0.64 mm²). In contrast, cells on PDMS Channel textures spread, proliferated, aligned along the channel axis, and created colonies that were more dense, and with lengths of longest colony axes that were significantly longer (3252 m) than those on the Smooth PDMS (1265 m) and control surfaces (1319 m). Cells on PDMS Channel textures were aligned at an angle of 14.44° relative to the channel axis, and the resulting colonies exhibited a significantly higher aspect ratio (13.72) compared to Smooth PDMS (1.57) and control surfaces (1.51).     

Death or illness of a family member,violence, interpersonal conflict,and financial difficulties as predictors of sickness absence: longitudinal cohort study on psychological and behavioral links

OBJECTIVE: Although an association between stressful life events and health problems has been demonstrated, the underlying mechanisms have remained unclear. We examined whether psychological problems and health-risk behaviors underpin the health effects of different event categories. METHOD: The initially healthy participants were 2991 (796 men, 2195 women) municipal employees who had taken no sick leave in 1995. In 1997, they completed a questionnaire requesting information on recent life events and psychological and behavioral factors. The outcome was recorded sickness absences in 1998. RESULTS: In men, the death or serious illness of a family member, violence, and financial difficulties increased the risk of later sickness absence. According to structural equation modeling, violence and financial difficulties also induced psychological problems such as anxiety, mental distress, and lowered sense of coherence. Psychological problems were associated with heightened cigarette and alcohol consumption, which in turn increased sickness absence. A corresponding structural model did not fit the data in relation to death or serious illness of a family member. In women, life events were associated with psychological problems and smoking but not sickness absence. CONCLUSIONS: Longitudinal evidence suggests that increased psychological problems and behaviors involving risk to health partially mediate the effect of stressful life events on health, as indicated by sickness absence. This model received support among men and for the event categories of violence and financial difficulties. Women were less affected by stressful life events than men.     

Optical rotation and linear and circular depolarization rates in diffusively scattered light from chiral,racemic, and achiral turbid media

The polarization properties of light scattered in a lateral direction from turbid media were studied. Polarization modulation and synchronous detection were used to measure, and Mueller calculus to model and derive, the degrees of surviving linear and circular polarization and the optical rotation induced by turbid samples. Polystyrene microspheres were used as scatterers in water solutions containing dissolved chiral, racemic, and achiral molecules. The preservation of circular polarization was found to exceed the linear polarization preservation for all samples examined. The optical rotation induced increased with the chiral molecule concentration only, whereas both linear and circular polarizations increased with an increase in the concentrations of chiral, racemic, and achiral molecules. This latter effect was shown to stem solely from the refractive index matching mechanism induced by the solute molecules, independent of their chiral nature.