The amino acid/K symporters for neutral amino acids along the midgut of lepidopteran larvae: Functional differentiations

The functional properties of the K⁺-dependent symporter for neutral amino acids have been investigated in brush border membrane vesicles prepared from the anterior, middle and posterior portions corresponding to the three morphologically distinguishable regions of the midgut of Bombyx mori larvae. An intravesicular accumulation of leucine was driven by a K⁺-gradient in the three preparations, but vesicles from the posterior tract displayed much higher uptake and accumulation values. Kinetic analysis of leucine uptake, performed in experimental conditions which mimic as closely as feasible experimentally those occurring in vivo (Δψ = −90 mV, pH_in7.2/pH_out8.7, [K⁺]_out100 mM), evidenced that the affinity for the amino acid was similar along the midgut (150 μM), but V_max in the posterior region was more than 11-fold higher than that of the anterior-middle tract (11.3 ± 0.7 and 0.98 ± 0.07 nmol/7s/mg protein, respectively). Leucine uptake was remarkably influenced by extravesicular pH and by Δψ only in vesicles from the posterior midgut: a lowering of pH to 7.2 caused a sevenfold increase of K_m, whereas in the absence of Δψ, V_max decreased threefold. The selectivity sequence for the alkali cations was somewhat different in the two midgut regions, but K⁺ remained the most effective. In the posterior midgut, the selectivity for K⁺ was greatly enhanced when a transmembrane electrical potential was present. Leucine kinetics as a function of external potassium concentration was hyperbolic in the posterior and sigmoidal in the anterior-middle part. Inhibition of leucine uptake induced by a 20-fold excess of different amino acids suggested the presence in both midgut tracts of a broad specificity system for neutral amino acids, with many-but not all-features in common with the B^o system of mammal intestinal and renal epithelial brush borders. However, there are differences between the two midgut regions as regard to the ability of the symporters to recognize the different amino acids, which concern the side chain and the presence of the aromatic ring. Altogether these data suggest that two kinds of symporters for neutral amino acids, with different functional properties, are expressed in the anterior-middle and posterior regions of the lepidopteran midgut.     

Probing the active sites and mechanisms of rat metalloproteases meprin A and B

Meprin A and B are highly regulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain alpha and beta subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physico-chemical factors. Homo-oligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homo-oligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.     

Activation of glutamate ionotropic connections of sensorimotor cortex neurons during conditioning

Changes in conditioned impulse reactions of neurons in sensorimotor cortex were studied during microiontophoretic application of glutamatergic and GABA ergic agonistic and antagonistic drugs. It was shown that ionotropic glutamate receptors (AMPA and NMDA) are activated by a conditioned stimulus. Not only large pyramidal neurons of deep cortical layers but surrounding short-axon inhibitory interneurons are involved in the reaction. It was shown that the activity of pyramidal neurons is under a constant inhibitory control from surrounding interneurons. This inhibition is involved in organization of excitatory cortical responses during conditioning.     

Engineering and monitoring cellular barrier models

Epithelia and endothelia delineate tissue compartments and control their environments by regulating the passage of ions and solutes. This barrier function is essential for the development and maintenance of multicellular organisms, and its dysfunction is associated with numerous human diseases. Recent advances in biomaterials and microfabrication technologies have evolved in vitro approaches for modelling biological barriers. Current microphysiological systems have become more efficient and reliable in mimicking the cell microenvironment. Additionally, methods for the quantification of barrier permeability have long provided significant insight into their underlying mechanisms. In this review, we outline the current techniques to quantify the barrier function of engineered tissues, and we also give an overview of recent microphysiological systems of biological barriers that emulate the microenvironment and microarchitecture of native tissues.     

Meprin proteolytic complexes at the cell surface and in extracellular spaces

Meprins are metalloproteinases of the astacin family and metzincin superfamily that are composed of evolutionarily related alpha and beta subunits, which exist as homo- and hetero-oligomeric complexes. These complexes are abundant at the brush border membranes of kidney proximal tubule cells and epithelial cells of the intestine, and are also expressed in certain leucocytes and cancer cells. Meprins cleave bioactive peptides such as gastrin, cholecystokinin and parathyroid hormone, cytokines such as osteopontin and monocyte chemotactic peptide-1, as well as proteins such as gelatin, collagen IV, fibronectin and casein. Database predictions and initial data indicate that meprins are also capable of shedding proteins, including itself, from the cell surface. Membrane-bound meprin subunits are composed of dimeric meprin beta subunits or tetrameric hetero-oligomeric alpha beta complexes of approx. 200-400 kDa, and can be activated at the cell surface; secreted forms of homo-oligomeric meprin alpha are zymogens that form high-molecular-mass complexes of 1-6 MDa. These are among the largest extracellular proteases identified thus far. The latent (self-associating) homo-oligomeric complexes can move through extracellular spaces in a non-destructive manner, and deliver a concentrated form of the metalloproteinase to sites that have activating proteases, such as sites of inflammation, infection or cancerous growth. Meprins provide examples of novel ways of concentrating proteolytic activity at the cell surface and in the extracellular milieu, which may be critical to proteolytic function.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.