Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices

In nested collagen matrices, human fibroblasts migrate from cell-containing dermal equivalents into surrounding cell-free outer matrices. Time-lapse microscopy showed that in addition to cell migration, collagen fibril flow occurred in the outer matrix toward the interface with the dermal equivalent. Features of this flow suggested that it depends on the same cell motile machinery that normally results in cell migration. Collagen fibril flow was capable of producing large-scale tissue translocation as shown by closure of a approximately 1-mm gap between paired dermal equivalents in floating, nested collagen matrices. Our findings demonstrate that when fibroblasts interact with collagen matrices, tractional force exerted by the cells can couple to matrix translocation as well as to cell migration.     

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the ¹⁴C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.     

Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains

Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle cells surrounding blood vessels, in hair follicles, and on adipocytes. Cells bearing the integrins alpha1 beta1 and alpha2 beta1 attach and spread on recombinant collagen XVI. Furthermore, collagen XVI induces the recruitment of these integrins into focal adhesion plaques, a principal step in integrin signaling. Of potential physiological relevance, these integrin-collagen XVI interactions may connect cells with specialized fibrils, thus contributing to the organization of fibrillar and cellular components within connective tissues. In cell-free binding assays, collagen XVI is more avidly bound by alpha1 beta1 integrin than by alpha2 beta1 integrin. Both integrins interact with collagen XVI via the A domain of their alpha subunits. A tryptic collagen XVI fragment comprising the collagenous domains 1-3 is recognized by alpha1 beta1 integrin. Electron microscopy of complexes of alpha1 beta1 integrin with this tryptic collagen XVI fragment or with full-length collagen XVI revealed a unique alpha1 beta1 integrin-binding site within collagen XVI located close to its C-terminal end.     

Refinement of Pig Retroperfusion Technique: Global Retroperfusion with Ligation of the Azygos Connection Preserves Hemodynamic Function in an Acute Infarction Model in Pigs (Sus scrofa domestica)

In ischemic hearts, venous retroperfusion is a potential myocardial revascularization strategy. This study aimed to refine the technical and functional aspects of a pig model of acute myocardial infarction and retroperfusion with respect to the azygos connection. Global retroperfusion after ligation of the ramus interventricularis paraconalis (equivalent to the left anterior descending artery in humans) was performed in 16 Landrace pigs (Sus scrofa domestica). Coronary sinus perfusion was performed in 8 pigs (P+) but not in the other 8 (P–), and the azygos vein was ligated (L+) 4 of the 8 pigs in each of these groups but left open (L–) in the remaining animals. Hemodynamic performance (for example, cardiac output, stroke volume) was significantly better in P+L+ pigs that underwent coronary sinus perfusion with ligation of the azygos vein compared with all other animals. In addition, troponin I release was significant lower in P+L+ pigs (1.7 ± 1.3 ng/mL) than in P–L– (5.47 ± 2.1 ng/mL), P–L+ (6.63 ± 2.4 ng/mL), and P+L– (4.81 ± 2.3 ng/mL) pigs. Effective retrograde flow and thus hemodynamic stability was achieved by ligation of the azygos vein. Therefore, experiments focusing on global retroperfusion will benefit from effective inhibition of the blood flow through the azygos vein.Abbreviations: ACS, aorta-to-coronary–sinus shunt, CS, coronary sinus, L, ligation, LAD, left anterior descending artery, P, perfusionAnimal models are used frequently to investigate myocardial revascularization techniques, and researchers have studied global or selective venous retroperfusion in dogs,²² pigs,⁹ and sheep.³³ The goal underlying retrograde coronary sinus (CS) perfusion is perfusion of the ischemic myocardium proximal to the occlusion or stenosis. This method frequently is used for delivering cardioplegic solutions during cardiac surgery. In addition, both clinical^{2,3,6,16,28,30} and experimental ^{11,21,25,34,37,42} studies have validated the efficiency of CS retroperfusion.Interpreting the results from experimental animal models and follow-up examinations of patients who have undergone venous revascularization has led to controversy.^9,37 In particular, technical problems with some studies have been identified. Previous animal studies on interspecies anatomic differences in mammals^4,7,19 have concentrated on the venous connections of the vessels draining the myocardium and have demonstrated a need for further feasibility studies of the pig model (German Landrace pigs, Sus scrofa domestica) that focus on hemodynamic performance.We wanted to characterize in detail the contribution of the azygos vein connection in swine during retroperfusion after myocardial infarction and hypothesized that ligation of the azygos vein would preserve hemodynamic function after ligation of the left anterior descending artery (LAD) in a global retroperfusion model in pigs.     

Galactocerebroside biosynthesis pathways of Mycoplasma species: an antigen triggering Guillain–Barré–Stohl syndrome

Infection by Mycoplasma pneumoniae has been identified as a preceding factor of Guillain–Barré–Stohl syndrome. The Guillain–Barré–Stohl syndrome is triggered by an immune reaction against the major glycolipids and it has been postulated that M. pneumoniae infection triggers this syndrome due to bacterial production of galactocerebroside. Here, we present an extensive comparison of 224 genome sequences from 104 Mycoplasma species to characterize the genetic determinants of galactocerebroside biosynthesis. Hidden Markov models were used to analyse glycosil transferases, leading to identification of a functional protein domain, termed M2000535 that appears in about a third of the studied genomes. This domain appears to be associated with a potential UDP-glucose epimerase, which converts UDP-glucose into UDP-galactose, a main substrate for the biosynthesis of galactocerebroside. These findings clarify the pathogenic mechanisms underlining the triggering of Guillain–Barré–Stohl syndrome by M. pneumoniae infections.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

<Emphasis Type="Italic">Daphnia</Emphasis>species diversity in Kenya,and a key to the identification of their ephippia

The distribution of Daphniaspecies in tropical Africa is poorly known and understood. Daphniaare assumed rare in tropical regions, but systematic studies covering large areas are sparse. We sampled the active community (live zooplankton) and/or the dormant community (diapausing egg banks in the sediment) of 41 standing water bodies in Kenya in search for Daphnia.Overall the dormant communities yielded 11 species of Daphnia, a species richness more than twice the species richness found in the active communities. Dormant community species diversity better reflects the spatial, and particularly the temporal (multi-annual) variation in environmental conditions available to Daphniain these tropical standing waters. Hence, we suggest that the dormant community be taken into account when assessing local zooplankton diversity, especially in fluctuating tropical lake ecosystems, where the presence of each local Daphniaspecies in the active community may be strongly seasonal or erratic. Geographic distribution data from this study are supplemented with previous records of Daphniain East Africa to provide an overview of the known distribution of Daphniain Kenya and neighbouring countries. We also present a detailed key for morphological identification of the ephippia of the 11 Daphniaspecies encountered, complemented with photographs and drawings of diagnostic characters.     

Phosphorylation of Gephyrin in Hippocampal Neurons by Cyclin-dependent Kinase CDK5 at Ser-270 Is Dependent on Collybistin

Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.