Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Two-level evolution of foraging agent communities

This paper presents simulation results of artificial foraging agent communities. The goal of each agent in the community is to find food. Once a food source is found, agents eat portions of it and carry some other portions to the nest (in a manner similar to ants) until the food is depleted. Agents may also communicate food positions when they are near each other. They are given a set of genes that control several characteristics, such as their activity, memory, scepticism, lying, etc. These genes are recombined and propagated by means of sexual reproduction. When one nest is superpopulated with agents, it can break in two nests. Agents can communicate only with those belonging to the same nest, which gives rise to emergent situations of competition and cooperation between the agents in the same nest, as well as competition between different nests. Other emergent phenomena such as the propagation of rumours are also studied.     

Linking cranial kinematics,buccal pressure,and suction feeding performance in largemouth bass

The rate and magnitude of buccal expansion are thought to determine the pattern of water flow and the change in buccal pressure during suction feeding. Feeding events that generate higher flow rates should induce stronger suction pressure and allow predators to draw prey from further away. We tested these expectations by measuring the effects of prey capture kinematics on suction pressure and the effects of the latter on the distance from which prey were drawn-termed suction distance. We simultaneously, but not synchronously, recorded 500-Hz video and buccal pressure from 199 sequences of four largemouth bass, Micropterus salmoides, feeding on goldfish. From the video, we quantified several kinematic variables associated with the head and jaws of the feeding bass that were hypothesized to affect pressure. In a multiple regression, kinematic data accounted for 79.7% of the variation among strikes in minimum pressure. Faster mouth opening and hyoid depression were correlated with lower pressures, a larger area under the pressure curve, and a faster rate of pressure reduction. In contrast, buccal pressure variables explained only 16.5% of the variation in suction distance, and no single pressure variable had a significant relationship with suction distance. Thus, although expected relationships between head kinematics and buccal pressure were confirmed, suction distance was only weakly related to buccal pressure. Three explanations are considered. First, bass may not attempt to maximize the distance from which prey are drawn. Second, the response of prey items to suction-induced flow depends on prey behavior and orientation and is, therefore, subject to considerable variation. Third, previous theoretical work indicates that water velocity decays exponentially with distance from the predator's mouth, indicating that variation among strikes in flow at the mouth opening is compressed away from the mouth. These findings are consistent with other recent data and suggest that suction distance is a poor metric of suction feeding performance.     

The coiled coil region (amino acids 129-250) of the tumor suppressor protein adenomatous polyposis coli (APC). Its structure and its interaction with chromosome maintenance region 1 (Crm-1)

The APC (adenomatous polyposis coli) tumor suppressor protein has many different intracellular functions including a nuclear export activity. Only little is known about the molecular architecture of the 2843-amino acid APC protein. Guided by secondary structure predictions we identified a fragment close to the N-terminal end, termed APC-(129-250), as a soluble and protease-resistant domain. We solved the crystal structure of APC-(129-250), which is monomeric and consists of three alpha-helices forming two separate antiparallel coiled coils. APC-(129-250) includes the nuclear export signal NES-(165-174) at the C-terminal end of the first helix. Surprisingly, the conserved hydrophobic amino acids of NES-(165-174) are buried in one of the coiled coils and are thus not accessible for interaction with other proteins. We demonstrate the direct interaction of APC-(129-250) with the nuclear export factor chromosome maintenance region 1 (Crm-1). This interaction is enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC-(129-250), which deletes two amino acids forming two of the major interhelical interactions within the coiled coil. These observations hint to a regulatory mechanism of the APC nuclear export activity by NES masking.     

Downregulation of the Petunia hybrida alpha-expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs

The expansins comprise a family of proteins that appear to be involved in the disruption of the noncovalent bonds between cellulose microfibrils and cross-linking glycans, thereby promoting wall creep. To understand better the expansion process in Petunia hybrida (petunia) flowers, we isolated a cDNA corresponding to the PhEXP1 alpha-expansin gene of P. hybrida. Evaluation of the tissue specificity and temporal expression pattern demonstrated that PhEXP1 is preferentially expressed in petal limbs during development. To determine the function of PhEXP1, we used a transgenic antisense approach, which was found to cause a decrease in petal limb size, a reduction in the epidermal cell area, and alterations in cell wall morphology and composition. The diminished cell wall thickness accompanied by a reduction in crystalline cellulose indicates that the activity of PhEXP1 is associated with cellulose metabolism. Our results suggest that expansins play a role in the assembly of the cell wall by affecting either cellulose synthesis or deposition.     

Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus

Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.     

Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B

The carboxy-terminus of ATP7B, the protein defective in the copper-transport disorder Wilson disease, was investigated with respect to its role in copper delivery to the ferroxidase ceruloplasmin. We use yeast as a model system to assess the functional capabilities of ATP7B variants. The yeast ferroxidase, Fet3p, acquires copper from Ccc2p and cannot function if Ccc2p is impaired; expression of wild-type ATP7B in ccc2 yeast complements the iron-deficient phenotype. Our results demonstrate that the C-terminus of ATP7B is necessary for protein stability, as removal of the nonmembranous terminus leads to reduced protein levels and cessation of growth in iron-limited medium. Growth is partially restored when an additional three amino acids are present and is near wild-type levels when only one-third of the C-terminus is present. Measurement of ferroxidase activity is a more sensitive indicator of copper transport function and allowed identification of impaired variants not detected with the growth assay.