Propulsive adaptation to changing gait speed

Understanding propulsion and adaptation to speed requirements is important in determining appropriate therapies for gait disorders. We hypothesize that adaptations for changing speed requirements occur primarily at the hip. The slow, normal and fast gait of 24 healthy young subjects was analyzed. The linear power was analyzed at the hip joint. The anterior-posterior and vertical induced accelerations of the hip were also determined. Linear power and anterior-posterior-induced acceleration (IA) analyses of the hip reveal that the lower limb joint's moments contribute to body forward propulsion primarily during late swing and early stance. Propulsive adaptations to speed changes occur primarily at the hip and secondarily at the ankle. These analyses show that hip muscles, particularly the hip extensors, are critical to propulsion. They also show that ankle function is primarily for support, but is important to propulsion, especially at slow speeds.     

Retinal GABA(A) receptors participate in the regulation of circadian responses to light

A role for retinal gamma-aminobutyric acid Type A (GABA(A)) receptors in the regulation of circadian responses to light was examined. Intraocular injections of the GABA(A) antagonist, bicuculline, were performed during the early (Circadian Time [CT] 13.5) and late subjective night (CT 20), followed by a light pulse. Bicuculline significantly decreased the magnitude of phase delays induced by light to 65%, whereas it had no effect on phase advances. To explore the nature of the inhibition elicited by bicuculline, an intensity-response curve was performed. Intraocular injections of bicuculline inhibited phase delays only when induced by high-saturating light illuminances (20 and 100 lux). No effect was observed at light intensities < or = 5 lux. These results suggest that retinal GABA(A) receptors modulate the responsivity of the circadian system to light.     

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.     

The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.     

Quantitative RT-PCR assay for VEGF mRNA in human tumors of the kidney

Angiogenesis is the formation of new capillaries from pre-existing vessels, and recent evidence has demonstrated that tumor growth is controlled mainly by angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor which is strongly angiogenic in vitro and in vivo. We have developed a quantitative RT-PCR assay for the measurement of VEGF mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The assay performance of this method in terms of practicability and reliability is reported with results that seem promising for its widespread use in the clinical laboratory. The method has been applied to the measurement of mRNA of VEGF in human renal cell carcinomas (RCC). Preliminary results show a significantly higher VEGF mRNA expression (ratio values between 181 and 2222) in tumor specimens compared to non-adjacent, non-tumoral tissue of the same subjects.     

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor- (TNF-) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg·kg^–1·day^–1 and 1.0 mg·kg^–1·day^–1) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF- and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF- and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity. apoptosis; cytokines; myosin heavy chains     

Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.     

The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).