Kinematic and kinetic factors that correlate with improved knee flexion following treatment for stiff-knee gait

Stiff-knee gait is a movement abnormality in which knee flexion during swing phase is significantly diminished. This study investigates the relationships between knee flexion velocity at toe-off, joint moments during swing phase and double support, and improvements in stiff-knee gait following rectus femoris transfer surgery in subjects with cerebral palsy. Forty subjects who underwent a rectus femoris transfer were categorized as "stiff" or "not-stiff" preoperatively based on kinematic measures of knee motion during walking. Subjects classified as stiff were further categorized as having "good" or "poor" outcomes based on whether their swing-phase knee flexion improved substantially after surgery. We hypothesized that subjects with stiff-knee gait would exhibit abnormal joint moments in swing phase and/or diminished knee flexion velocity at toe-off, and that subjects with diminished knee flexion velocity at toe-off would exhibit abnormal joint moments during double support. We further hypothesized that subjects classified as having a good outcome would exhibit postoperative improvements in these factors. Subjects classified as stiff tended to exhibit abnormally low knee flexion velocities at toe-off (p<0.001) and excessive knee extension moments during double support (p=0.001). Subjects in the good outcome group on average showed substantial improvement in these factors postoperatively. All eight subjects in this group walked with normal knee flexion velocity at toe-off postoperatively and only two walked with excessive knee extension moments in double support. By contrast, all 10 of the poor outcome subjects walked with low knee flexion velocity at toe-off postoperatively and seven walked with excessive knee extension moments in double support. Our analyses suggest that improvements in stiff-knee gait are associated with sufficient increases in knee flexion velocity at toe-off and corresponding decreases in excessive knee extension moments during double support. Therefore, while stiff-knee gait manifests during the swing phase of the gait cycle, it may be caused by abnormal muscle activity during stance.     

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.     

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

A large T cell invagination with CD2 enrichment resets receptor engagement in the immunological synapse

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.     

Helminths in Mesaspis monticola (Squamata: Anguidae) from Costa Rica, with the description of a new species of Entomelas (Nematoda: Rhabdiasidae) and a new species of Skrjabinodon (Nematoda: Pharyngodonidae)

Entomelas duellmani n. sp. (Rhabditida: Rhabdiasidae) from the lungs and Skrjabinodon cortagoensis n. sp. (Oxyurida: Pharyngodonidae) from the intestines of Mesaspis monticola (Sauria: Anguidae) are described and illustrated. E. duellmani is the sixth species assigned to the genus and is the third species described from the Western Hemisphere. It is easily separated from other neotropical species in the genus by pre-equatorial position of its vulva. Skrjabinodon cartagoensis is the 24th species assigned to the genus and differs from other neotropical species in the genus by female tail morphology.     

Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex

Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. The enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B2 bradykinin receptor (B2R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). In agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B2R-/- mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35-40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis.     

Resistance of three co-occurring resprouter Erica species to highly frequent disturbance

The resistance to experimental, highly frequent disturbance has been analysed in three congeneric, strong-resprouter species (Erica australis, E. scoparia and E. arborea) that co-occur in heath-dominated communities of the northern side of the Strait of Gibraltar, southern Spain. To do so, mature individuals of the three species from a long undisturbed location were clipped at the ground level every sixth month during two years. The relationship between the resprouted biomass dry weight (as indicative of the resprouting vigour) and the upper surface area of the lignotuber along the experiment was established separately for each species at each clipping event by means of linear regressions analysis. The resprouting vigour of the three species was compared by means of independent one-way ANOVAs within each clipping event. Resprouting vigour decreased after recurrent clippings in the three species. Nevertheless, significant differences between species in this loss of resprouting vigour were detected, being E. scoparia the most resistant to the experimental, highly frequent clipping. It is concluded that experimental levels of recurrent disturbance may help to find out differences in resilience within similar (taxonomically, morfologically and/or ecologically), strong-resprouter plant species. Considering the history of forestry management in the nothern side of the Strait of Gibraltar, differences in this regard between the three Erica species may contribute to explain their somewhat segregated ecological distribution in this region.