In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus

Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.     

NHERF2/SIP-1 interacts with mouse SRY via a different mechanism than human SRY

In mammals, male sex determination is controlled by the SRY protein, which drives differentiation of the bipotential embryonic gonads into testes by activating the Sertoli cell differentiation program. The morphological effects of SRY are well documented; however, its molecular mechanism of action remains unknown. Moreover, SRY proteins display high sequence variability among mammalian species, which makes protein motifs difficult to delineate. We previously isolated SIP-1/NHERF2 as a human SRY-interacting protein. SIP-1/NHERF2, a PDZ protein, interacts with the C-terminal extremity of the human SRY protein. Here we showed that the interaction of SIP-1/NHERF2 and SRY via the SIP-1/NHERF2 PDZ1 domain is conserved in mice. However, the interaction occurs via a domain that is internal to the mouse SRY protein and involves a different recognition mechanism than human SRY. Furthermore, we show that mouse and human SRY induce nuclear accumulation of the SIP-1/NHERF2 protein in cultured cells. Finally, a transgenic mouse line expressing green fluorescent protein under the control of the mouse Sry promoter allowed us to show that SRY and SIP-1/NHERF2 are co-expressed in the nucleus of pre-Sertoli cells during testis determination. Taken together, our results suggested that the function of SIP-1/NHERF2 as an SRY cofactor during testis determination is conserved between human and mouse.     

Effect of locally applied drugs on the pH of luminal fluid in the endolymphatic sac of guinea pig

The aim of the present work was to assess the effect of various drugs applied locally on the pH of the luminal fluid (pH(lum)) in guinea pig endolymphatic sac. pH(lum) and transepithelial potential, when measured in vivo by means of double-barrelled pH-sensitive microelectrodes, were 7.06 +/- 0.08 and +6.1 +/- 0.34 mV (mean +/- SE; n = 84), respectively, which is consistent with a net acid secretion in the luminal fluid of the endolymphatic sac. Bafilomycin and acetazolamide increased and decreased, respectively, pH(lum). Amiloride, ethylisopropylamiloride, ouabain, and Schering 28080 had no effect on pH(lum). Results obtained with inhibitors of anionic transport systems were inconclusive; e.g., DIDS reduced pH(lum), whereas neither SITS nor triflocin had any effect. We conclude that bafilomycin-sensitive H(+)-ATPase activity accounts for the transepithelial acid gradient measured in the endolymphatic sac and that intracellular and membrane-bound carbonic anhydrase probably participates in regulating endolymphatic sac pH(lum). The relationship between acid pH, endolymph volume, and Ménière's disease remains to be further investigated.     

Integrin alpha(IIb)beta3 signals lead cofilin to accelerate platelet actin dynamics

Cofilin, in its Ser3 dephosphorylated form, accelerates actin filament turnover in cells. We report here the role of cofilin in platelet actin assembly. Cofilin is primarily phosphorylated in the resting platelet as evidenced by a specific antibody directed against its Ser3 phosphorylated form. After stimulation with thrombin under nonstirring conditions, cofilin is reversibly dephosphorylated and transiently incorporates into the actin cytoskeleton. Its dephosphorylation is maximal 1–2 min after platelet stimulation, shortly after the peak of actin assembly occurs. Cofilin rephosphorylation begins 2 min after activation and exceeds resting levels by 5–10 min. Cofilin is dephosphorylated with identical kinetics but fails to become rephosphorylated when platelets are stimulated under stirring conditions. Cofilin is normally rephosphorylated when platelets are stimulated in the presence of Arg-Gly-Asp-Ser (RGDS) peptide or wortmannin to block _IIb3 cross-linking and signaling or in platelets isolated from a patient with Glanzmann thrombasthenia, which express only 2–3% of normal _IIb3 levels. Furthermore, actin assembly and Arp2/3 complex incorporation in the platelet actin cytoskeleton are decreased when _IIb3 is engaged. Our results suggest that cofilin is essential for actin dynamics mediated by outside-in signals in activated platelets.     

Enhanced dermal and retinal vascular permeability in streptozotocin-induced type 1 diabetes in Wistar rats: blockade with a selective bradykinin B1 receptor antagonist

The vascular complications associated with type 1 diabetes are to some extent related to the dysfunction of the endothelium leading to an increased vascular permeability and plasma extravasation in the surrounding tissues. The various micro- and macro-vascular complications of diabetes develop over time, leading to nephropathy, retinopathy and neuropathy and cardiomyopathy. In the present study, the effect of a novel selective bradykinin B1 receptor (BKB1-R) antagonist, R-954, was investigated on the changes of vascular permeability in the skin and retina of streptozotocin (STZ)-induced type 1 diabetic rats. Plasma extravasation increased in the skin and retina of STZ-diabetic rats after 1 week and persisted over 4 weeks following STZ injection. Acute treatment with R-954 (2 mg/kg, bolus s.c.) highly reduced the elevated vascular permeability in both 1- and 4-week STZ-diabetic rats. These results showed that the inducible BKB1-R subtype modulates the vascular permeability of the skin and retina of type 1 diabetic rats and suggests that BKB1-R antagonists could have a beneficial role in diabetic neuropathy and retinopathy.     

Sampling frequencies and measurement error for linear and temporal gait parameters in primate locomotion

Quantitative analyses of animal motion are increasingly easy to conduct using simple video equipment and relatively inexpensive software packages. With careful use, such analytical tools have the potential to quantify differences in movement between individuals or species and to allow insights into the behavioral consequences of morphological differences between taxa. However, as with any other type of measurement, there are errors associated with kinematic measurements. Because normative kinematic data on human and nonhuman primate locomotion are used to model aspects of gait of fossil hominins, errors in the extant data influence the accuracy of fossil gait reconstructions. The principal goal of this paper is to illustrate the effect of camera speeds (frame rates) on kinematic measurement errors, and to demonstrate how these errors vary with subject size, movement velocity, and sample size. Kinematic data for human walking and running (240 Hz), as well as data for primate quadrupedal walking and running (180 Hz) were used as inputs for a simulation of the measurement errors associated with various linear and temporal kinematic variables. Measurement errors were shown to increase as camera speed, subject body size, and interval duration all decrease, and as movement velocity increases. These results have implications for the methods used to calculate subject velocity and suggest that using a moving marker to measure the linear displacements of the body is preferable to the use of a stationary marker. Finally, while slower camera speeds will always result in higher measurement errors than do faster camera speeds, this effect can be moderated to some extent by collecting sufficiently large samples of data.