Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes.

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed 'epithelial-membrane glycoprotein' (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.     

Differential Roles of the Pseudomonas aeruginosa PA14 rpoN Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.     

Predicting the success of an invader: Niche shift versus niche conservatism

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.     

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes

Summary The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E₁ both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha₁-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha, and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.Abbreviations cAMP Adenosine-3,5-Cyclic Monophosphate - cGMP Guanosine-3,5-Cyclic Monophosphate - G_i, G_S Guanine nucleotide-binding proteins linked to inhibition and stimulation, respectively, of adenylate cyclase - GTP Guanosine-5-triphosphate - PDE Cyclic Nucleotide Phosphodiesterase - PGE₁ Prostaglandin E₁     

Phosphorylations of cyclin-dependent kinase 2 revisited using two-dimensional gel electrophoresis

To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.     

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.     

WIDESPREAD HOST‐DEPENDENT HYBRID UNFITNESS IN THE PEA APHID SPECIES COMPLEX

Linking adaptive divergence to hybrid unfitness is necessary to understand the ecological factors contributing to reproductive isolation and speciation. To date, this link has been demonstrated in few model systems, most of which encompass ecotypes that occupy relatively early stages in the speciation process. Here we extend these studies by assessing how host‐plant adaptation conditions hybrid fitness in the pea aphid, Acyrthosiphon pisum. We made crosses between and within five pea aphid biotypes adapted to different host plants and representing various stages of divergence within the complex. Performance of F1 hybrids and nonhybrids was assessed on a “universal” host that is favorable to all pea aphid biotypes in laboratory conditions. Although hybrids performed equally well as nonhybrids on the universal host, their performance was much lower than nonhybrids on the natural hosts of their parental populations. Hence, hybrids, rather than being intrinsically deficient, are maladapted to their parents’ hosts. Interestingly, the impact of this maladaptation was stronger in certain hybrids from crosses involving the most divergent biotype, suggesting that host‐dependent postzygotic isolation has continued to evolve late in divergence. Even though host‐independent deficiencies are not excluded, hybrid maladaptation to parental hosts supports the hypothesis of ecological speciation in this complex.     

Two-week longitudinal survey of bone architecture alteration in the hindlimb-unloaded rat model of bone loss: sex differences

The goal of this study was to determine, through a longitudinal follow-up, whether sex influences bone adaptation during simulated weightlessness. Twelve-week-old male and female Wistar rats were hindlimb unweighted for 2 wk, and the time course of bone alteration was monitored in vivo by means of densitometry and unbiased three-dimensional quantitative microcomputed tomography at 7 and 14 days. Compared with male rats, female rats had twice more cancellous bone volume at the proximal tibia at baseline, and this bone volume continued to increase, whereas in males it stabilized. Conversely, cortical area was greater in males than in females, and in both sexes cortical bone was still expanding. Hindlimb unloading resulted in larger reductions in males than in females in both cortical and cancellous compartments. In females, trabecular thickness and number decreased mildly, whereas in males trabecular number was dramatically reduced. In both sexes, the trabecular network became less connected and more rod-like shaped. Bone cellular activities evaluated by histomorphometry showed decreased bone formation rate in both sexes and increased resorption activity only in males. In conclusion, in female rats unloaded-related cancellous alterations reversed the growing process, whereas in males, which show lower growth process, it induced an accentuation of age-related cancellous bone changes for most of the parameters.