Identification of a host-associated species complex using molecular and morphometric analyses,with the description of Gyrodactylus rugiensoides n. sp. (Gyrodactylidae,Monogenea)

Gyrodactylus rugiensis was originally described as a parasite occurring on the marine gobies Pomatoschistus minutus and Pomatoschistus microps. In our preliminary survey this species was also frequently found on Pomatoschistus pictus and Pomatoschistus lozanoi. Subsequent molecular analysis of the internal transcribed spacers rDNA region revealed that this parasite actually represents a complex of two apparently cryptic species, one restricted to P. microps and the other shared by P. minutus, P. lozanoi and P. pictus. Morphometric analyses were conducted on 17 features of the opisthaptoral hard parts of specimens collected from all four host species. Standard discriminant analysis showed a clear separation of both genotypes by significant differences in marginal hook and ventral bar features. Statistical classifiers (linear discriminant analysis and nearest neighbours) resulted in an estimated misclassification rate of 4.7 and 3.1%, respectively. Based on molecular, morphological and statistical analyses a new species, Gyrodactylus rugiensoides is described. This species seems to display a lower host-specificity than generally observed for Gyrodactylus species as it infects three sympatric host species.However, seasonal and host-dependent morphometric variation is shown for G. rugiensoides collected on P. pictus. Host-switching and gene flow might be important factors preventing speciation on closely related and sympatric host species. The presence of host associated species complexes in this Gyrodactylus-Pomatoschistus system is also confirmed by the presence of two host-dependent genotypes within G. micropsi found on P. minutus and P. lozanoi, and P. microps, respectively. By comparing host and parasite phylogeny, phylogenetic and ecological factors influencing host-specificity are discussed.     

Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts

Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.     

Effects of autonomic disruption and inactivity on venous vascular function

The effects of autonomic disruption and inactivity were studied on the venous vascular system. Forty-eight subjects, 24 with spinal cord injury (SCI) and 12 sedentary and 12 active able-bodied controls, participated in this study. Peripheral autonomic data were obtained to estimate sympathetic vasomotor control [low-frequency component of systolic blood pressure (LF(SBP))]. Vascular parameters were determined using strain-gauge venous occlusion plethysmography: venous capacitance (VC), venous emptying rate (VER), and total venous outflow (VO(t)). An additional vascular parameter was calculated: venous compliance [(VC/occlusion pressure) x 100]. VC and VO(t) were significantly different (SCI < sedentary < active). VER adjusted for VC was not different for any group comparison, whereas venous compliance was significantly lower in the SCI group than in the able-bodied groups and in the sedentary group compared with the active group. Regression analysis for the total group revealed a significant relationship between LF(SBP) and venous compliance (r = 0.64, P < 0.0001). After controlling for LF(SBP) through analysis of covariance, we found that mean differences for all venous vascular parameters did not change from unadjusted mean values. Our findings suggest that in subjects with SCI, the loss of sympathetic vasomotor tone contributes more than inactivity to reductions in venous vascular function. Heightened VC, VO(t), vasomotor tone, and venous compliance in the active group compared with the sedentary group imply that regular endurance training contributes to optimal venous vascular function and peripheral autonomic integrity.     

Lack of effects of glycineB receptor ligands on the psychostimulant-induced discriminative stimuli in rats

To examine the role of glycineB receptors in the stimulus effects induced by psychostimulants, separate groups of rats were trained to discriminate amphetamine (AMPH; 1 mg/kg) from saline (SAL), or cocaine (COC; 10 mg/kg) from SAL, using a two-lever operant procedure. Substitution studies showed that neither 1-aminocyclopropanecarboxylic acid (ACPC; 200 mg/kg) nor 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-(H)quinolone (L-701,324; 3 mg/kg), being a partial agonist or an antagonist at glycineB receptors, respectively, generalized for the training drugs. Combination tests of glycineB ligands demonstrated that injection of a fixed dose of ACPC (200 mg/kg) or L-701,324 (3 mg/kg) together with different doses of AMPH or COC practically did not modify dose-response curves of the psychostimulants, nor did it affect their ED50 values. Our results indicate that glycineB receptors do not play a role in the discriminative effects of AMPH and COC.     

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.     

Matrix Metalloproteinases -8 and -9 and Tissue Inhibitor of Metalloproteinase-1 in Burn Patients. A Prospective Observational Study

IntroductionMatrix metalloproteinases (MMPs) -8 and -9 are released from neutrophils in acute inflammation and may contribute to permeability changes in burn injury. In retrospective studies on sepsis, levels of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) differed from those of healthy controls, and TIMP-1 showed an association with outcome. Our objective was to investigate the relationship between these proteins and disease severity and outcome in burn patients.MethodsIn this prospective, observational, two-center study, we collected plasma samples from admission to day 21 post-burn, and burn blister fluid samples on admission. We compared MMP-8, -9, and TIMP-1 levels between TBSA<20% (N = 19) and TBSA>20% (N = 30) injured patients and healthy controls, and between 90-day survivors and non-survivors. MMP-8, -9, and TIMP-1 levels at 24-48 hours from injury, their maximal levels, and their time-adjusted means were compared between groups. Correlations with clinical parameters and the extent of burn were analyzed. MMP-8, -9, and TIMP-1 levels in burn blister fluids were also studied.ResultsPlasma MMP-8 and -9 were higher in patients than in healthy controls (P<0.001 and P = 0.016), but only MMP-8 differed between the TBSA<20% and TBSA>20% groups. MMP-8 and -9 were not associated with clinical severity or outcome measures. TIMP-1 differed significantly between patients and controls (P<0.001) and between TBSA<20% and TBSA>20% groups (P<0.002). TIMP-1 was associated with 90-day mortality and correlated with the extent of injury and clinical measures of disease severity. TIMP-1 may serve as a new biomarker in outcome prognostication of burn patients.