Rabbit polymorphonuclear leukocyte migration in vivo in response to lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella

Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes in response to injected LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella. A salmonella LPS was used as reference endotoxin. No differnece in chemotactic activity between the Veillonella LPS and LPS from Salmoneila was found. Fusobacterium LPS whoed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bacteroides LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per microliter of exudate was found.     

Growth cone navigation in substrate-bound ephrin gradients

Graded distributions of ephrin ligands are involved in the formation of topographic maps. However, it is still poorly understood how growth cones read gradients of membrane-bound guidance molecules. We used microcontact printing to produce discontinuous gradients of substrate-bound ephrinA5. These consist of submicron-sized protein-covered spots, which vary with respect to their sizes and spacings. Growth cones of chick temporal retinal axons are able to integrate these discontinuous ephrin distributions and stop at a distinct zone in the gradient while still undergoing filopodial activity. The position of this stop zone depends on both the steepness of the gradient and on the amount of substrate-bound ephrin per unit surface area. Quantitative analysis of axon outgrowth shows that the stop reaction is controlled by a combination of the local ephrin concentration and the total amount of encountered ephrin, but cannot be attributed to one of these parameters alone.     

Low-intensity two-step absorption of chlorophyll A in vivo.

The intensity-dependent transmission of primary leaves of Triticum aestivum seedlings at lambda = 694 nm was measured with single pulses of a Q-switch ruby laser. At photon flux densities above 2 x 10(17) cm-2s-1 a decrease of transmission was observed. The result is interpreted as a two-step absorption of cooperative units of 10(5)-10(6) chlorophyll molecules.     

Mechanisms of plant response to gradients and after disturbances

Plant responses to gradients and after disturbances are important because they help define key ecosystem functions, such as productivity and decomposition. In this article I review studies that have tended to examine these influences on plants separately, presenting an incomplete view of vegetation dynamics. I then present a conceptual model of interaction between gradients and disturbance focusing on the mechanisms of plant-plant replacement, which largely determine species composition, abundance, and dispersion and are often mediated by changes in resource availability. The model consists of mechanisms that are either interactive and dependent on the presence of the replacing species or noninteractive and driven by the relationship between changing resource conditions in patches and the life histories / ecotolerances of species. The model explores variation in mechanism strength among different disturbances defined primarily by severity (e.g., amount of biomass lost), among gradients that differ in temperature and precipitation, and among species of fundamentally differing life histories. The main assumption is that plants mainly respond to the most limited resource in a given gradient × disturbance situation, as dictated by the relative severity of the disturbance versus the relative severity of the gradient. Finally, I sketch a general theory of plant responses containing the testable hypotheses from the model and also including axioms, assumptions, and deductions arising from these components.

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Resumen  Las condiciones pos disturbios son importantes porque ayudan a definir las claves del funcionamiento de un ecosistema tales como la productividad y la descomposición. En este artículo repaso estudios anteriores, que han tendido a estudiar la influencia de factures en las planta de manera separada, presentando una visión incompleta de la dinámica de la vegetación. En este estudio presento un modelo conceptual de las interaciones entre los gradientes naturales y los disturbios enfocado en los mecanismos de reemplazo planta-planta, los cuales influyen grandemente la composición, abundacia, y disperción de especies en un bosque y usualmente están mediados por cambios en la disponibilidad de recursos. El modelo consiste de mecanismos que interaccionan y dependen de la presencia de reemplazamiento de especies o no interaccionan y están dirigidos por la interrelatión entre cambios en la disponibilidad de recursos en parches y la historia de vida / ecotolerancia de las especies. El modelo explora variaciones en la fuerza del mecanismo entre diferentes disturbios definidos por la severidad (e.g., perdida de biomas), entre gradientes que se diferencia en temperature y presipitación, y entre especies con distintos historias de vida. El planteamiento principal es que las plantas responden mayormente al recurso más limitado en determinado gradiente multiplicado por la situación de disturbio correspondiente, según lo dicta la severidad relativa del disturbio contra la severidad relativa del gradiente. Finalmente, formulo una teoría general de la respuesta de las plantas que contiene la hipótesis comprobable del modelo e incluye axiomas, asunciones, y deducciones que surjen de los componentes.

     

Selectins and glycosyltransferases in leukocyte rolling in vivo

Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified - P-, E- and L-selectin - which recognize and bind to crucial carbohydrate determinants on selectin ligands. Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo conditions. Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level. However, high endothelial venule-expressed selectin ligands were instrumental in uncovering the crucial role of post-translational modifications for selectin ligand activity. Several glycosyltransferases, such as core 2 beta1,6-N-acetylglucosaminyltransferase-I, beta1,4-galactosyltransferases, alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins. In addition, modifications, such as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands.     

Auditory stimulus intensity gradients and response to methylphenidate in ADD children

Using an auditory stimulus intensity paradigm, we obtained both event related potentials (ERPs) and press and release reaction times (RT) from a large sample of children with Attention Deficit Disorder (ADD). The ERP gradients to three tone intensities were used to classify the children as augmenters (steep gradients), moderates, or reducers (shallow or negative gradients). The RT data were used to classify the children as strong or sensitive, following neo-Pavlovian guidelines. The children were then cross-classified on these two dimensions and compared on cognitive, behavioral, and performance measures. The groups were also compared in response to two dosage levels of methylphenidate. Based on prior studies, we hypothesized that: 1. ERP augmenters would respond as well to the low as high dose but that reducers would respond better to high than low dose; and II. sensitive types (RT measure) would do better on the higher dose and strong types on the low dose. The first hypothesis was confirmed on a performance task but not on behavioral ratings. At the lower dose, augmenters improved most and reducers least on a 10-minute coding task presumed to require sustained attention. There was no support for the second hypothesis either in ratings or performance. The ERP augmentation measure was significantly related to teacher rated attentiveness; i.e., reducers and moderates were rated more adversely. The RT sensitivity measure tended to be related to achievement; i.e., strong types had lower reading and spelling scores. The ERP and RT sensitivity measures were not significantly correlated.     

Integrating individual search and navigation behaviors in mechanistic movement models

Understanding complex movement behaviors via mechanistic models is one key challenge in movement ecology. We built a theoretical simulation model using evolutionarily trained artificial neural networks (ANNs) wherein individuals evolve movement behaviors in response to resource landscapes on which they search and navigate. We distinguished among non-oriented movements in response to proximate stimuli, oriented movements utilizing perceptual cues from distant targets, and memory mechanisms that assume prior knowledge of a target??s location and then tested the relevance of these three movement behaviors in relation to size of resource patches, predictability of resource landscapes, and the occurrence of movement barriers. Individuals were more efficient in locating resources under larger patch sizes and predictable landscapes when memory was advantageous. However, memory was also frequently used in unpredictable landscapes with intermediate patch sizes to systematically search the entire spatial domain, and because of this, we suggest that memory may be important in explaining super-diffusion observed in many empirical studies. The sudden imposition of movement barriers had the greatest effect under predictable landscapes and temporarily eliminated the benefits of memory. Overall, we demonstrate how movement behaviors that are linked to certain cognitive abilities can be represented by state variables in ANNs and how, by altering these state variables, the relevance of different behaviors under different spatiotemporal resource dynamics can be tested. If adapted to fit empirical movement paths, methods described here could help reveal behavioral mechanisms of real animals and predict effects of anthropogenic landscape changes on animal movement.     

A novel phospholipase D2-Grb2-WASp heterotrimer regulates leukocyte phagocytosis in a two-step mechanism

Phagocytosis is a primary innate response of both macrophages and neutrophils involving the formation of filamentous actin (F-actin)-rich protrusions that are extended around opsonized pathogens to form a phagocytic cup, resulting in their subsequent internalization. The molecular mechanism for this is still not completely understood. We now show for the first time that phospholipase D2 (PLD2) binds to growth factor receptor-bound protein 2 (Grb2) and to the Wiskott-Aldrich syndrome protein (WASp) to form a heterotrimer complex, PLD2-Grb2-WASp, and present the mechanism of interaction. Grb2 binds to the Y169/Y179 residues of PLD2 using its only SH2 domain, and it interacts with the poly-proline region of WASp using its two SH3 domains. The PLD2-Grb2-WASp heterotrimer can be visualized in early phagocytic cups of macrophages ingesting opsonized red blood cells, where it associates with polymerized actin. Cup colocalization and phagocytosis are disrupted with mutants that alter binding at either of the two proteins or by silencing Grb2 with RNA interference (RNAi). WASp association to PLD2-K758R, a lipase-inactive mutant, still occurs, albeit at lower levels, indicating that PLD2 plays a second role in phagocytosis, which is the production of phosphatidic acid (PA) and activation of phosphatidylinositol 5-kinase (PI5K) with subsequent synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)). The latter can be blocked with RNAi, which negates phagocytosis. Lastly, a constitutively "open" active form of WASp (WASp-L270P) brings phagocytosis to its maximum level, which can be mimicked with WASp-WT plus PLD2 or plus PA. Since neither a protein-protein disruption nor lack of PLD activity completely negates cup formation or phagocytosis, we posit a two-step mechanism: PLD2 anchors WASp at the phagocytic cup through Grb2 following protein-protein interactions and also activates it, making key lipids available locally. The heterotrimer PLD2-Grb2-WASp then enables actin nucleation at the phagocytic cup and phagocytosis, which are at the center of the innate immune system function.     

OMSSAGUI: An open-source user interface component to configure and run the OMSSA search engine

We here present a user-friendly and extremely lightweight tool that can serve as a stand-alone front-end for the Open MS Search Algorithm (OMSSA) search engine, or that can directly be used as part of an informatics processing pipeline for MS driven proteomics. The OMSSA graphical user interface (OMSSAGUI) tool is written in Java, and is supported on Windows, Linux, and OSX platforms. It is an open source under the Apache 2 license and can be downloaded from http://code.google.com/p/mass-spec-gui/.     

Microbial response to environmental gradients in a ceramic-based diffusion system

A solid, porous matrix was used to establish steady-state concentration profiles upon which microbial responses to concentration gradients of nutrients or antimicrobial agents could be quantified. This technique relies on the development of spatially defined concentration gradients across a ceramic plate resulting from the diffusion of solutes through the porous ceramic matrix. A two-dimensional, finite-element numerical transport model was used to predict the establishment of concentration profiles, after which concentration profiles of conservative tracers were quantified fluorometrically and chemically at the solid-liquid interface to verify the simulated profiles. Microbial growth responses to nutrient, hypochloride, and antimicrobial concentration gradients were then quantified using epifluorescent or scanning confocal laser microscopy. The observed microbial response verified the establishment and maintenance of stable concentration gradients along the solid-liquid interface. These results indicate the ceramic diffusion system has potential for the isolation of heterogeneous microbial communities as well as for testing the efficacy of antimicrobial agents. In addition, the durability of the solid matrix allowed long-term investigations, making this approach preferable to conventional gel-stabilized systems that are impeded by erosion as well as expansion or shrinkage of the gel.     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

A search for strain differences in response of mice to mutagenesis by thio-TEPA

After treatment of mice with thio-TEPA Malashenko and colleagues found differences among inbred strains in yield of dominant lethals and of chromosome aberrations in bone marrow, which they attributed to genes affecting repair. An attempt was made to confirm this work by comparing yields of dominant lethals in different strains of females mated to the same strain of males. However, no differences were found, all strain combinations giving 42-49% dominant lethals after a dose of 2 mg/kg thio-TEPA to late spermatids. Thus, the existence of genetic differences in repair of thio-TEPA induced lesions between strains CBA and C57BL/6J and between C3H/He and 101/H is not confirmed. Possible reasons for the discrepant results are discussed.     

Factors influencing human leukocyte adherence in vivo

The effects of different factors on leukocyte adherence was evaluated by using human peripheral blood before and after treatment (application). Magnesium ions, anti-inflammatory drugs and dextran have decreased neutrophil adherence. Calcium ions, propranolol, blood transfusion caused a significant decrease in adherence. These findings support the conclusion that the adherence test, which is influenced by numerous factors and whose results had a wide scatter, is not a useful tool in clinical diagnosis.     

Periodicity and chaos in the response of Halobacterium to temporal light gradients

Halobacteria spontaneously reverse their swimming direction about every 10 s. This periodicity can be altered by light stimuli. We found that temporal exponential changes in light intensity, depending on wavelength and sign, lengthened or shortened the intervals between reversals. Within a limited range of steepness, light gradients enforced a new stable periodicity upon the system. Outside this range, they caused period doubling or induced a sequence of reversal events without any obvious regularity. An analysis of a functional relationship between apparently irregular periods by plotting each period as a function on the preceding one yielded a clearly discernible non-random structure, which shows some similarities to the one obtained by a model calculation for a periodically perturbed limit cycle oscillator. These results indicate that external forcing of the system may generate chaos. When the decay of intracellular sensory signals is delayed by inhibition of protein methylation the transition from periodic to aperiodic behavior occurs at a lower steepness of the gradient. We therefore assume that the generation of either periodic or deterministic chaotic behavior is determined by the relation between the signal lifetime and the frequency of stimulus inputs. The strong indications for transitions from periodic to chaotic behavior can be regarded as a further support of our hypothesis that the behavioral pattern of Halobacterium is controlled by an endogeneous oscillator.     

Metabolic response of polymorphonuclear leukocyte to arachidonic and linoleic acids

Various stimuli act on polymorphonuclear leukocytes (PMN), activating membrane-bound phospholipase A2 and C, and diglyceride lipase and then liberating unsaturated fatty acids (USFAs). These liberated USFAs are immediately metabolized through various metabolic pathways such as cyclooxygenase, lipoxygenase, phosphatidylinositol metabolism etc. It is possible that the metabolic intermediates of these pathways reveal various physiological actions. This work was undertaken to clarify whether stimuli on PMN depend on these USFAs themselves or on their oxidation products. The following results were obtained: 1. USFAs such as arachidonate and linoleate stimulate PMN, accelerating superoxide (O2) generation, depolarization of membrane potential and increase in [Ca2+]i. 2. Oxidation products of USFAs have no stimulative effect on PMN. The decrease in the stimulative effect of these USFAs following their oxidation is proportional to the quantitative decrease in non-oxidized linoleate. 3. USFAs accelerate membrane permeability of Ca2+, and their oxidation products enhance non-specific membrane permeability in proportion to the formation of monohydroxy compound. These results suggest that stimulative effects of USFAs on PMN do not depend on their oxidation products but on unoxidized fatty acids. Furthermore, among the oxidation products of the USFAs, monohydroxy compound acts as a strong perturber of membrane and accelerates membrane permeability.     

Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients

Preliminary studies have shown that asymmetric transbilayer distributions of phosphatidic acid (PA) can be induced by transmembrane pH gradients (delta pH) in large unilamellar vesicles [Hope et al. (1989) Biochemistry 28, 4181-4187]. Here the mechanism of PA transport is examined employing TNS as a fluorescent probe of lipid asymmetry. It is shown that the kinetics of PA transport are consistent with the transport of the uncharged (protonated) form. Transport of the neutral form can be rapid, exhibiting half-times for transbilayer transport of approximately 25 s at 45 degrees C. It is also shown that PA transport is associated with a large activation energy (28 kcal/mol) similar to that observed for phosphatidylglycerol. The maximum induced transbilayer asymmetry of PA corresponded to approximately 95% on the inner monolayer for vesicles containing 5 mol % PA.