The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins

The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.     

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (K_d = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.     

Exercise-induced lipid mobilization in subcutaneous adipose tissue is mainly related to natriuretic peptides in overweight men

Involvement of sympathetic nervous system and natriuretic peptides in the control of exercise-induced lipid mobilization was compared in overweight and lean men. Lipid mobilization was determined using local microdialysis during exercise. Subjects performed 35-min exercise bouts at 60% of their maximal oxygen consumption under placebo or after oral tertatolol [a beta-adrenergic receptor (AR) antagonist]. Under placebo, exercise increased dialysate glycerol concentration (DGC) in both groups. Phentolamine (alpha-AR antagonist) potentiated exercise-induced lipolysis in overweight but not in lean subjects; the alpha(2)-antilipolytic effect was only functional in overweight men. After tertatolol administration, the DGC increased similarly during exercise no matter which was used probe in both groups. Compared with the control probe under placebo, lipolysis was reduced in lean but not in overweight men treated with the beta-AR blocker. Tertatolol reduced plasma nonesterified fatty acids and insulin concentration in both groups at rest. Under placebo or tertatolol, the exercise-induced changes in plasma nonesterified fatty acids, glycerol, and insulin concentrations were similar in both groups. Exercise promoted a higher increase in catecholamine and ANP plasma levels after tertatolol administration. In conclusion, the major finding of our study is that in overweight men, in addition to an increased alpha(2)-antilipolytic effect, the lipid mobilization in subcutaneous adipose tissue that persists during exercise under beta-blockade is not dependent on catecholamine action. On the basis of correlation findings, it seems to be related to a concomitant exercise-induced rise in plasma ANP when exercise is performed under tertatolol intake and a decrease in plasma insulin.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.     

Ser and Thr Residues Modulate the Conformation of Pro-Kinked Transmembrane α-Helices

Functionally required conformational plasticity of transmembrane proteins implies that specific structural motifs have been integrated in transmembrane helices. Surveying a database of transmembrane helices and the large family of G-protein coupled receptors we identified a series of overrepresented motifs associating Pro with either Ser or Thr. Thus, we have studied the conformation of Pro-kinked transmembrane helices containing Ser or Thr residues, in both g+ and g− rotamers, by molecular dynamics simulations in a hydrophobic environment. Analysis of the simulations shows that Ser or Thr can significantly modulate the deformation of the Pro. A series of motifs, such as (S/T)P and (S/T)AP in the g+ rotamer and the TAP and PAA(S/T) motifs in the g− rotamer, induce an increase in bending angle of the helix compared to a standard Pro-kink, apparently due to the additional hydrogen bond formed between the side chain of Ser/Thr and the backbone carbonyl oxygen. In contrast, (S/T)AAP and PA(S/T) motifs, in both g+ and g−, and PAA(S/T) in g+ rotamers decrease the bending angle of the helix by either reducing the steric clash between the pyrrolidine ring of Pro and the helical backbone, or by adding a constrain in the form of a hydrogen bond in the curved-in face of the helix. Together with a number of available experimental data, our results strongly suggest that association of Ser and Thr with Pro is commonly used in transmembrane helices to accommodate the structural needs of specific functions.     

Comparative Genomics of Insect-Symbiotic Bacteria: Influence of Host Environment on Microbial Genome Composition

Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.     

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone–DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal ‘tail’ of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an ‘epitope’ consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Structure of the accessory reproductive glands of the male migratory grasshopper,Melanoplus sanguinipes

The accessory reproductive glands of Melanoplus sanguinipes comprise two bilateral masses of 16 tubules each, distinguishable in sexually mature insects as four white, ten short hyaline, one long hyaline, and a seminal vesicle. Over most of its length, the wall of each tubule consists of a simple glandular epithelium resting on a basal lamina, surrounded by a thin layer of circular muscle. However, near the junction with the ejaculatory duct, the wall of each tubule has a much thickened circular muscle layer and squamous or cuboidal epithelium, the region serving to regulate movement of secretion into the ejaculatory duct. Interdigitation of adjacent epithelial cells is common, and several kinds of specialized junctions occur. In the glandular region, all epithelial cells appear the same and may be flattened, cuboidal, or columnar depending on the tubule type. Except for those of the seminal vesicle, the glandular epithelial cells share ultrastructural features typical of cells engaged in the synthesis of protein for export. Despite these general similarities, in most instances subtle differences occur in the cellular ultrastructure of the epithelia of each tubule and in the appearance of their luminal secretions, suggesting that the tubules are functionally specialized.