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.     

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.     

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.     

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.     

Human impact on vegetation at the Alpine tree-line ecotone during the last millennium: lessons from high temporal and palynological resolution

Three mires and a small lake in the Swiss and Austrian Alps were studied palynologically at high resolution, covering the last 1,000, 400, 50 and 1,200 years, respectively. Methodological lessons include: (1) Sub-decadal resolution in upper, little-decomposed peat layers reveals recurrent marked fluctuations in both percentages and influx of regional tree-pollen types, reflecting variations in pollen production rather than in plant-population sizes. (2) Intermittent, single-spectrum pollen maxima in samples of sub-decadal resolution indicate pollen transport in clumps. This type of pollen transport may remain unrecognized in sections with lower sampling resolution, which may then lead to inappropriate interpretation in terms of plant-population sizes. (3) The detection of short-lived phases of human impact in decomposed peat requires sampling intervals as close as 0.2 cm. (4) PAR (pollen influx) may reflect vegetation dynamics more faithfully than percentages. Reliable PAR, however, is difficult to achieve in Alpine mires due to past human impact on peat growth, even when complex depth–age modelling techniques are used. Critical comparison of PAR with percentages is therefore essential. (5) Careful consideration of spatial scales in pollen signals (local–regional and subdivisions) is essential for a realistic palaeo-ecological interpretation. Results in terms of past human impact on vegetation are summarized as follows: (1) Trends in pollen types reflecting regional human action are in general agreement with earlier findings for the western Swiss Alps, allowing for regional differences. (2) All mires in the Alps investigated here and in an earlier study experienced human impact during the last millennium. The studied small lake, lying in sub-alpine pasture, records forest dynamics at a lower elevation since a.d. 800.     

Isolation of a rat mitochondrial release factor. Accommodation of the changed genetic code for termination

A single release factor has been isolated and partially purified from rat mitochondria. It requires ethanol in addition to the specific termination codon when assayed in a heterologous system with Escherichia coli ribosomes. The factor recognizes the codons UAA and UAG but not UGA, and therefore it has been designated mtRF-1. A factor of the bacterial RF-2 type, which in E. coli recognizes UGA, or of the mammalian type, which recognizes all three termination codons, has not been detected in mitochondria. The absence of a factor responding to UGA accommodates the use of this codon as a signal for tryptophan in the rat mitochondrial genetic code. The mtRF-1 could translate all of the known termination codons in the rat mitochondrial genome. It does not respond to AGG and AGA which in bovine and human mitochondrial DNA code for termination but which in rat mitochondria may not code for either an amino acid or for termination.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.