Mechanical properties of shock-absorbing pylons used in transtibial prostheses

Prosthetic manufacturers have developed shock-absorbing pylons to attenuate the transient forces of foot-ground contact in order to supplement the residual capacity of lower limb amputees. The purpose of this study was to measure the elastic and damping properties of two frequently prescribed pylons (the ICON Shock Pylon and the Mercury TT Pyramid Pylon) at frequencies enveloping those observed during gait using pseudo-static compressive and dynamic cyclic testing methods. Results showed that the spring constants were linear functions of deformation (ranging from 74 to 110 N/mm and 91 to 157 N/mm for the ICON and the TT Pylons, respectively) while the damping force was a function of the square root of velocity combined with a coulomb element (1.6x0.5 + 21 and 7.4x0.5 + 102 N for the ICON and the TT Pylon, respectively).     

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey (Petromyzon marinus)

gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.     

In vivo measurement of coronary circulation angiotensin-converting enzyme activity in humans

Angiotensin-converting enzyme (ACE) is present on the luminal surface of the coronary vessels, mostly on capillary endothelium. ACE is also expressed on coronary smooth muscle cells and on plaque lipid-laden macrophages. Excessive coronary circulation (CC)-ACE activity might be linked to plaque progression. Here we used the biologically inactive ACE substrate (3)H-labeled benzoyl-Phe-Ala-Pro ([(3)H]BPAP) to quantify CC-ACE activity in 10 patients by means of the indicator-dilution technique. The results were compared with atherosclerotic burden determined by coronary angiography. There was a wide range of CC-ACE activity as revealed by percent [(3)H]BPAP hydrolysis (30-74%). The atherosclerotic extent scores ranged from 0.0 to 66.97, and the plaque area scores ranged from 0 to 80 mm(2). CC-ACE activity per unit extracellular space (V(max)/K(m)V(i)), an index of metabolically active vascular surface area, was correlated with myocardial blood flow (r = 0.738; P = 0.03) but not with measures of the atherosclerotic burden. These results show that CC-ACE activity can be safely measured in humans and that it is a good marker of the vascular area of the perfused myocardium. It does not, however, reflect epicardial atherosclerotic burden, suggesting that local tissue ACE may be more important in plaque development.     

Endocannabinoids and related fatty acid derivatives in pain modulation

The brain produces at least five compounds that possess sub-micromolar affinity for cannabinoid receptors: anandamide, 2-arachidonoylglycerol, noladin ether, virodhamine, and N-arachidonoyldopamine (NADA). One function of these and/or related compounds is to suppress pain sensitivity. Much evidence supports a role of endocannabinoids in pain modulation in general, and some evidence points to the role of particular endocannabinoids. Related endogenous fatty acid derivatives such as oleamide, palmitoylethanolamide, 2-lineoylglycerol, 2-palmitoylglycerol, and a family of arachidonoyl amino acids may interact with endocannabinoids in the modulation of pain sensitivity.     

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration-dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of alpha-synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in alpha-synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.     

Molecular cloning and characterization of glucanase inhibitor proteins: coevolution of a counterdefense mechanism by plant pathogens

A characteristic plant response to microbial attack is the production of endo-beta-1,3-glucanases, which are thought to play an important role in plant defense, either directly, through the degradation of beta-1,3/1,6-glucans in the pathogen cell wall, or indirectly, by releasing oligosaccharide elicitors that induce additional plant defenses. We report the sequencing and characterization of a class of proteins, termed glucanase inhibitor proteins (GIPs), that are secreted by the oomycete Phytophthora sojae, a pathogen of soybean, and that specifically inhibit the endoglucanase activity of their plant host. GIPs are homologous with the trypsin class of Ser proteases but are proteolytically nonfunctional because one or more residues of the essential catalytic triad is absent. However, specific structural features are conserved that are characteristic of protein-protein interactions, suggesting a mechanism of action that has not been described previously in plant pathogen studies. We also report the identification of two soybean endoglucanases: EGaseA, which acts as a high-affinity ligand for GIP1; and EGaseB, with which GIP1 does not show any association. In vitro, GIP1 inhibits the EGaseA-mediated release of elicitor-active glucan oligosaccharides from P. sojae cell walls. Furthermore, GIPs and soybean endoglucanases interact in vivo during pathogenesis in soybean roots. GIPs represent a novel counterdefensive weapon used by plant pathogens to suppress a plant defense response and potentially function as important pathogenicity determinants.     

The effect of host size on the sex ratio of Syngaster lepidus, a parasitoid of Eucalyptus longhorned borers (Phoracantha spp.)

The solitary larval ectoparasitoid, Syngaster lepidus Brullé, parasitizes the cryptic larvae of two wood-boring beetles, Phoracantha recurva Newman and Phoracantha semipunctata F. The objective of this study was to determine how the female parasitoids allocated the sex of progeny when presented with larval hosts of uniform size classes. Host size was directly correlated with age of the Phoracantha larval hosts. Groups of Phoracantha larvae of a single age class (2-, 3-, 4-, or 5-week-old) were exposed to parasitoids, and sex ratios of the resulting parasitoid progeny from each host age class were determined. A significant relationship was observed among the sizes of P. recurva and P. semipunctata hosts and the sex ratio of emerging parasitoids. Parasitized 2-week-old beetle larvae of both Phoracantha spp. produced only male S. lepidus progeny, whereas older larval hosts produced increasing proportions of female parasitoids (up to 80% females from 5-week-old hosts). Two-week-old Phoracantha larvae of both species produced fewer parasitoids than host larvae 3–5-week-old. The size of parasitoid progeny consistently increased with host larval age (size), and female parasitoids were larger than males across all host size classes. Male S. lepidus developed in approximately 25 days from 2-week-old hosts, and 19–21 days in 3–5-week-old hosts. Female S. lepidus developed in 22–25 days, with developmental time increasing with host size.     

Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases

The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.