Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

Seasonal Biomass and Carbohydrate Allocation Patterns in Southern Minnesota Curlyleaf Pondweed Populations

Four southern Minnesota populations of curlyleaf pondweed ( Potamogeton crispus L.) were sampled monthly from January 2001 to November 2002 to determine seasonal phenological, biomass, and carbohydrate allocation patterns. Low periods of carbohydrate storage in the seasonal phenological cycle indicate potentially vulnerable periods in the plant’s life cycle and may be the ideal time to initiate management and control efforts.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Nutrient digestibility and growth response of rainbow trout (Salmo gairdneri ) fed different carbohydrate types

Seven groups of fingerling rainbow trout (S. gairdneri ) were fed for 10 weeks on 0%, 10%, 20% and 30% of cassava or rice in isonitrogenous diets. Optimum growth and food utilization was at 20% dietary cassava. High fiber content of the control diet did not suppress protein digestibility in this group. Rather, at all levels, protein digestibility was good and remained between 84.4% and 90.1%. However, in the control group, carbohydrate digestibility was very poor. The cassava diets which had the highest digestible energy as carbohydrate produced the best growth performance, food utilization and protein sparing. At the levels studied, the dietary carbohydrates produced no hyperglycamic effect on the fish

Single-cell analysis of the physiology of mechanosensation in bacteria

Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.

Dietary carbohydrate requirement of Heterobranchus longifilis

The dietary carbohydrate requirement of Heterobranchus longifilis was evaluated in two separate experiments.In the first experiment, varying levels of carbohydrate ranging from 28, 24 to58 72% were fed to the fish of mean weight 1.83~c0.02g. Results revealed that the polynomial regression curve for the mean weight gain and the carbohydrate levels did not present a point where Y-max is equal to X-max and so the requirement was not obtained. The second experiment was therefore, conducted with lower levels of carbohydrate ranging from 17.00 to 20.86% and fed to fish with mean weight 0.49~c0.02g. Based on growth and feed efficiency data the carbohydrate requirement was determined to be 19.5%

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Relação dos sistemas de reparo de DNA em Escherichia coli K-12 com resistência a antibióticos, características adesivas e formação de biofilme

As espécies reativas de oxigênio (ERO) são geradas durante o metabolismo celular normal e podem produzir vários danos oxidativos no DNA, tais como lesões nas bases nitrogenadas ou sítios apurínico/apirimidínico (AP). Essas lesões podem acarretar acúmulo de sítios de mutações, caso esses danos não sejam reparados. Entretanto, as bactérias possuem vários mecanismos de defesa contra as ERO que desempenham um importante papel na manutenção da fisiologia. O objetivo deste trabalho foi o de avaliar se sistemas enzimáticos, como o reparo por excisão de bases (BER), sistema SOS e SoxRS, interferem em respostas como a sensibilidade aos antibióticos, aderência das células bacterianas a superfícies bióticas ou abióticas e formação de biofilme. Os mutantes utilizados no presente estudo são todos derivados de Escherichia coli K-12 e os resultados obtidos mostraram que, dos mutantes BER testados, o único que apresentou diferença no perfil de sensibilidade aos antimicrobiamos em relação à cepa selvagem (AB1157) foi o mutante xthA- (BW9091), deficiente em exonuclease III. No teste de aderência qualitativo realizado com linhagem de células HEp-2 (originária de carcinoma de laringe humana) foi observado que onze cepas da nossa coleção, apresentaram um padrão denominando like-AA, contrastando com o que era esperado para as cepas de E. coli utilizadas como controle negativo, que apresentam aderência discreta sem padrão típico. A aderência manose-sensível via fímbria do tipo I avaliada nesse estudo mostrou que essa fimbria, possui um papel relevante na intensidade da aderência e filamentação nessas cepas estudas. A filamentação é uma resposta SOS importante para que o genoma seja reparado antes de ser partilhado pelas células filhas. Além disso, com relação à formação de biofilme, oito cepas apresentaram um biofilme forte sendo que essa resposta não foi acompanhada pelo aumento da intensidade de filamentação. Nossos resultados em conjunto sugerem o envolvimento de estresse oxidativo na definição de parâmetros como sensibilidade a antimicrobianos, padrão e intensidade de aderência, filamentação e formação de biofilme nas amostras de E. coli K-12 avaliadas neste trabalho. Sugerimos que a aderência gera estresse oxidativo causando danos no DNA, o que leva a indução do sistema SOS resultando na resposta de filamentação observada.

Comparative investigations on the respiratory physiology of euryhaline gammarids with special reference to salinity adaptation. [Translation from: Helgolaender Wissenschaftliche Meeresuntersuchungen 23(4) 485-534, 1972.]

Members of the family Gammaridae are very closely interrelated. There arises the question as to how far they also differ amongst themselves through physiological characteristics. Comparative respiratory and physiological experiments were made on the five euryhaline species Gammarus locusta, G. oceanicus, G. salinus, G. zaddachi and G. duebeni. The respiratory measurements carried out within the framework of this experiment were occupied with the relationships between oxygen consumption and body size depending on salinity. They also had the object of determing the variations in metabolic intensity after an abrupt change in the salt content of the external medium, and to establish the period of time for the process of adaptation. As the experiments were carried out polarographically in a testing plant with continuous flow-through, and the method which was applied permitted continuous recording over prolonged intervals, there could also be carried out comparisons between metabolism at rest and under activity, and the alterations of oxygen consumption during the process of moulting could be measured.

Physiology of nitrate reduction in Pseudomonas aeruginosa. [Translation from: Z.allg.Mikrobiol. 13, 83-94, 1973.]

Pseudomonas aeruginosa has for some time been known as a denitrifier. Pseudomonas aeruginosa was chosen for further studies, because P. aeruginosa occurs abundantly in Plusssee and moreover there are contradictory assertions on the gas products of denitrification by this bacteria. In experimental research the pattern of growth and gas production of Pseudomonas aeruginosa on nutrient broth was studied.

The Physiology and Computation of Pyramidal Neurons

A variety of neural signals have been measured as correlates to consciousness. In particular, late current sinks in layer 1, distributed activity across the cortex, and feedback processing have all been implicated. What are the physiological underpinnings of these signals? What computational role do they play in the brain? Why do they correlate to consciousness? This thesis begins to answer these questions by focusing on the pyramidal neuron. As the primary communicator of long-range feedforward and feedback signals in the cortex, the pyramidal neuron is set up to play an important role in establishing distributed representations. Additionally, the dendritic extent, reaching layer 1, is well situated to receive feedback inputs and contribute to current sinks in the upper layers. An investigation of pyramidal neuron physiology is therefore necessary to understand how the brain creates, and potentially uses, the neural correlates of consciousness. An important part of this thesis will be in establishing the computational role that dendritic physiology plays. In order to do this, a combined experimental and modeling approach is used.

This thesis beings with single-cell experiments in layer 5 and layer 2/3 pyramidal neurons. In both cases, dendritic nonlinearities are characterized and found to be integral regulators of neural output. Particular attention is paid to calcium spikes and NMDA spikes, which both exist in the apical dendrites, considerable distances from the spike initiation zone. These experiments are then used to create detailed multicompartmental models. These models are used to test hypothesis regarding spatial distribution of membrane channels, to quantify the effects of certain experimental manipulations, and to establish the computational properties of the single cell. We find that the pyramidal neuron physiology can carry out a coincidence detection mechanism. Further abstraction of these models reveals potential mechanisms for spike time control, frequency modulation, and tuning. Finally, a set of experiments are carried out to establish the effect of long-range feedback inputs onto the pyramidal neuron. A final discussion then explores a potential way in which the physiology of pyramidal neurons can establish distributed representations, and contribute to consciousness.

The effects of environmental stress on the physiology of growth in rainbow trout, Salmo gairderi Richardson

There is little doubt that both mammalian and teleost growth hormones can accelerate growth and increase food conversion efficiency in all commonly-reared species of salmonid fish. In those vertebrates that have been closely studied (predominantly mammals), the pituitary hormone somatotropin (GH or growth hormone) is a prime determinant of somatic growth. The hormone stimulates protein biosynthesis and tissue growth, enhances lipid utilization and lipid release from the adipose tissues (a protein-sparing effect) and suppresses the peripheral utilization of glucose. The present study is a prerequisite for future work on growth hormone physiology in salmonids and should contribute to our understanding of the mechanisms of growth suppression in stressed fish. Plasma growth hormone (GH) levels were measured in rainbow trout using a radioimmunoassay developed against chinook salmon growth hormone.

The ecological impact of different mechanisms of chronic sub-lethal toxicity on feeding and respiratory physiology

Sub-lethal toxicity tests, such as the scope-for-growth test, reveal simple relationships between measures of contaminant concentration and effect on respiratory and feeding physiology. Simple models are presented to investigate the potential impact of different mechanisms of chronic sub-lethal toxicity on these physiological processes. Since environmental quality is variable, even in unimpacted environments, toxicants may have differentially greater impacts in poor compared to higher quality environments. The models illustrate the implications of different degrees and mechanisms of toxicity in response to variability in the quality of the feeding environment, and variability in standard metabolic rate. The models suggest that the relationships between measured degrees of toxic stress, and the maintenance ration required to maintain zero scope-for-growth, may be highly nonlinear. In addition it may be possible to define critical levels of sub-lethal toxic effect above which no environment is of sufficient quality to permit prolonged survival.

Physiological Determinants of Invasive Success Linking Distribution Patterns to Metabolic Physiology in Native and Invasive Blue Mussels (genus Mytilus)

In the early 20th century, a blue mussel species from the Mediterranean invaded the California coast and subsequently out-competed the native species south of Monterey Bay. Like other invasive species, Mytilus galloprovincialis has physiological traits that make it successful in habitats formerly occupied by the native M. trossulus, namely its adaptation to warm sea surface temperatures. This study looks at the current genotype distributions and enzymatic activities of field-acclimatized mussels within the hybrid zone where the species co-occur as well as mussels that have been acclimated for four weeks to different temperature and salinity conditions. In the field-acclimatized and laboratory-acclimated mussels, the native species exhibited significantly higher enzyme rates, which may reflect an evolutionary adaptation to compensate to low habitat temperatures. Indeed, the results of the laboratory acclimation indicate that these differences are genetically based. Whether an acclimation capacity exists may require even longer-term acclimation to different temperatures. Current findings suggest that the further spread of the invasive species is likely to be governed in large measure by the potentially counteracting effects of rising temperatures, which would favor the northerly spread of M. galloprovincialis, and increased winter precipitation, which would favor the persistence of M. trossulus. However, the success of M. galloprovincialis during acclimation to ‘dilute’ salinity (25 ppt) suggests that the invasive species can tolerate a greater salinity range than previously thought. Thus, further investigation is needed to build a comprehensive predictive model of the movement of M. galloprovincialis and the hybrid zone along the California coast.