The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.     

Hyphal morphogenesis in Aspergillus nidulans

The formation of hyphae that grow solely by apical extension is a defining feature of filamentous fungi. Hyphal morphogenesis involves several key steps, including the establishment and maintenance of a stable polarity axis, as well as cell division via the deposition of septa. Several filamentous fungi have been employed in attempts to decipher the mechanisms underlying these steps. Amongst these fungi, Aspergillus nidulans has proven to be a particularly valuable model. The genetic tractability of this fungus coupled with the availability of sophisticated post-genomics resources has enabled the identification and characterization of numerous genes involved in hyphal morphogenesis. Here, we summarize current progress towards understanding the function of these genes and the mechanisms involved in polarized hyphal growth and septation in A. nidulans. We also highlight important areas for future investigation.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.     

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome‐wide association study

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome‐wide association study in an F₂ Charolais × German Holstein cross‐breed population to identify genetic variants that affect behaviour‐related traits assessed in an open‐field and novel‐object test and analysed their putative impact on milk performance. Of 37 201 tested single nucleotide polymorphism (SNPs), four showed a genome‐wide and 37 a chromosome‐wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel‐object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.     

Thyroxine-induced differential mortality of cleft lip mouse embryos: dose- and time-response studies of the A/WySn strain

Pregnant A/WySn mice, 20 to 30% of whose offspring have spontaneous cleft lip, were treated with thyroxine. Following treatment, cleft lip and normal embryos died, but cleft lip embryos died at a higher rate. The increased liability of cleft lip embryos to thyroxine-induced death was considered as a possible experimental route to identify the basic genetic defect that causes cleft lip. A time-response study indicated that cleft lip embryos responded more than normals following treatment on any of days 7 to 12 of gestation, that there is no sharply defined critical period, and that normal and cleft lip embryos do not differ in time of maximum sensitivity. A dose-response study showed linear responses of normal and cleft lip embryos on a probit-log dose scale, with a common slope and LD50's of 1.9 and 1.3 mg respectively. These dose-response properties indicate that normal and cleft lip embryos are probably killed by the same mechanism, but differ in dosage tolerance. That is, they differ quantitatively, not qualitatively. Thyroxine did not significantly change the cleft lip frequency, and the difference between normal and cleft lip embryos that leads to cleft lip itself is therefore not in the same pathway as that which leads to thyroxine-induced death. A hypothetical example of the defect basic to both pathways is presented.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.