LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.     

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction. cytoskeleton; prestress; stress fibers; mechanotransduction; mechanical deformation     

DNA analysis on fox faeces and competition induced niche shifts

Interference competition can force inferior competitors to change their distribution patterns. It is, however, possible that the dominant competitor poses a higher threat during certain times of the year, for example during reproduction. In such cases, the inferior competitor is expected to change its distribution accordingly. We used a molecular species identification method on faeces to investigate how the spatial overlap between arctic and red foxes changes between seasons. The results show that arctic and red foxes are sympatric during winter, but allopatric in summer as arctic foxes retreat to higher altitudes further from the tree-line during the breeding season.     

Positive health: connecting well-being with biology

Two key types of well-being, eudaimonic and hedonic, are reviewed. The first addresses ideas of self-development, personal growth and purposeful engagement, while the second is concerned with positive feelings such as happiness and contentment. How well-being varies by age and socio-economic standing is briefly summarized, followed by examination of its biological correlates (neuroendocrine, immune, cardiovascular, rapid eye movement (REM) sleep). Preliminary findings on a sample of ageing women showed that those with higher levels of eudaimonic well-being had lower levels of daily salivary cortisol, pro-inflammatory cytokines, cardiovascular risk, and longer duration REM sleep compared with those showing lower levels of eudaimonic well-being. Hedonic well-being, however, showed minimal linkage to biomarker assessments. Future research directions building on these initial findings are discussed.     

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites

In partially denervated rodent muscle, terminal Schwann cells (TSCs) located at denervated end plates grow processes, some of which contact neighboring innervated end plates. Those processes that contact neighboring synapses (termed "bridges") appear to initiate nerve terminal sprouting and to guide the growth of the sprouts so that they reach and reinnervate denervated end plates. Studies conducted prior to knowledge of this potential involvement of Schwann cells showed that direct muscle stimulation inhibits terminal sprouting following partial denervation (Brown and Holland, 1979). We have investigated the possibility this inhibition results from an alteration in the growth of TSC processes. We find that stimulation of partially denervated rat soleus muscle does not alter the length or number of TSC processes but does reduce the number of TSC bridges. Stimulation also reduces the number of TSC bridges that form between end plates during reinnervation of a completely denervated muscle. The nerve processes ("escaped fibers") that normally grow onto TSC processes during reinnervation are also reduced in length. Therefore, stimulation alters at least two responses to denervation in muscles: (1) the ability of TSC processes to form or maintain bridges with innervated synaptic sites, and (2) the growth of axons along processes extended by TSCs.     

Compound heterozygosity at the FMR1 gene

Individuals affected with Fragile X syndrome are usually characterized at the DNA level by the presence of at least 200 CGG repeats in the 5' untranslated region of the FMR1 gene; this number of repeats is defined as a full mutation. Repeats that number 50-200 usually define those with premutations and are termed unaffected carriers. We report here a compound heterozygous female who carried CGG repeats in the FMR1 gene that fall within the premutation and full mutation ranges. The former appears to have been inherited from the father, whereas the latter is an expansion of the premutation carried by the proband's mother. Therefore, the offspring of the proband will carry a significant risk of being affected with Fragile X syndrome, and the paternal uncle and any cousins should be counselled for being at risk for this syndrome.     

Early disruption of pregnancy as a manifestation of seasonal infertility in pigs

All gilts and sows in production from which the detailed production information was available in a 160-sow unit were included to the study. In winter-spring, there were complete data available from 47 animals and in summer-autumn from 64 animals. The farm had a consistent history of the seasonally reduced farrowing rate in summer-autumn. Success of inseminations was monitored during a 4-month breeding period in winter-spring and in summer-autumn. Each animal was bled twice a week for 6 weeks starting a day before insemination and the blood samples were assayed to determine serum progesterone concentration. The blood samples were also assayed for cortisol to detect any acute infectious response. Starting on day 18, animals were pregnancy tested by transcutaneous real time ultrasound twice a week. In winter-spring, the farrowing rate was 72% (58 inseminations, 1.2 inseminations/sow) and in summer-autumn 63% (81 inseminations, 1.3 inseminations/sow). In winter-spring, there was only one detected case of early disruption of pregnancy (EDP), whereas nine such cases were recognised in summer-autumn. Five out of those nine animals returned to oestrus with a mean insemination to oestrus interval of 25.8+/-1.6 days. One sow returned to oestrus 35 days after insemination and three sows did not return to oestrus within 45 days. However, two of these sows had progesterone profiles that indicated an undetected oestrus around day 25. In those nine animals, no acute phase infectious response as indicated by a rise in serum cortisol was evident. Serum progesterone concentrations in the animals eventually loosing the pregnancy tended to be lower on day 13 (no significant difference) and were significantly lower on day 20 when compared with animals remaining pregnant. There was no difference in serum progesterone levels of pregnant animals between winter-spring and summer-autumn. Litter size was not affected by the season. The weaning to oestrus interval tended to be longer in summer-autumn. This study showed that the seasonally decreased farrowing rate is partly caused by EDP. The lowered progesterone concentrations in summer-autumn were demonstrable only in "problem animals".     

Toward the automated solid-phase synthesis of oligoglucosamines: systematic evaluation of glycosyl phosphate and glycosyl trichloroacetimidate building blocks

Glucosamines are common components of many biologically important oligosaccharides. Reported is a systematic evaluation of glucosamine phosphates and trichloroacetimidates as glycosylating agents for the efficient construction of beta-(1 --> 6) glucosamine linkages. A set of differentially protected glucosamine donors incorporating a host of amine protecting groups, including 2-phthaloyl, benzyloxycarbonyl (Z), trichloroetheoxycarbonyl (Troc) and trichloroacetyl (TCA) protective groups, were prepared. Donors were initially evaluated for reactivity and protecting group compatibility in a solution-phase study with a model 6-hydroxyl galactose acceptor. Based on these results, glucosamine donor 10 was selected for the solution-phase synthesis of a beta-(1 --> 6)-glucosamine pentasaccharide. Finally, building block 10 proved well suited for use in the automated solid-phase synthesis of a repeating unit trisaccharide. An assessment of glucosamine phosphate donors as potential glycosylating agents for a variety of glucosamine linkages is also discussed.