Increased albumin plasma efflux contributes to hypoalbuminemia only during early phase of sepsis in rats

The mechanisms leading to hypoalbuminemia in sepsis were explored by measuring plasma volume, albumin distribution, plasma albumin transcapillary escape rate (TER), and efflux (TER x albumin intravascular pool). These parameters were quantified in infected rats, injected intravenously with live Escherichia coli, and pair-fed and well-fed rats using an injection of (35)S-albumin and measuring plasma and whole body albumin concentrations. Animals were studied on days 1, 6, and 10 after infection. In pair-fed rats, neither albumin distribution nor exchange rate between the intra- and extravascular compartments was modified. The increase of plasma volume after infection partly explained hypoalbuminemia. Infection resulted in a reduction of the total albumin pool of the body all along the experimental period, indicating a net loss of the protein. Albumin TER (%/day) was significantly increased 1 and 6 days after infection, but the absolute efflux was increased only on day 1. Normal values were observed on day 10. Therefore, an accelerated plasma efflux contributes to hypoalbuminemia only during the early period of sepsis. During this phase, the protein was retained in the extravascular space where it was probably catabolized. Later on, other factors are probably involved.     

Intraventricular melanin-concentrating hormone stimulates water intake independent of food intake

The lateral hypothalamus (LH) has a critical role in the control of feeding and drinking. Melanin-concentrating hormone (MCH) is an orexigenic peptidergic neurotransmitter produced primarily in the LH, and agouti-related protein (AgRP) is an orexigenic peptidergic neurotransmitter produced exclusively in the arcuate (ARC), an area that innervates the LH. We assessed drinking and eating after third ventricular (i3vt) administration of MCH and AgRP. MCH (2.5, 5, and 10 micro g i3vt) significantly increased food as well as water intake over 4 h when administered during either the light or the dark portion of the day-night cycle. When MCH (5 micro g) was administered to rats with access to water but no food, they drank significantly more water than when given the vehicle. AgRP (7 micro g i3vt), on the other hand, increased water intake but only in proportion to food intake during the dark and the light, and water intake was not increased after i3vt AgRP in the absence of food. Hence, in contrast to AgRP, MCH elicits increased water intake independent of food intake. These results are consistent with historical data linking activity of the LH with water as well as food intake.     

The osmoregulatory and the amino acid-regulated responses of system A are mediated by different signal transduction pathways

The osmotic response of system A for neutral amino acid transport has been related to the adaptive response of this transport system to amino acid starvation. In a previous study (Ruiz-Montasell, B., M. Gómez-Angelats, F.J. Casado, A. Felipe, J.D. McGivan, and M. Pastor-Anglada. 1994. Proc. Natl. Acad. Sci. USA. 91:9569-9573), a model was proposed in which both responses were mediated by different mechanisms. The recent cloning of several isoforms of system A as well as the elucidation of a variety of signal transduction pathways involved in stress responses allow to test this model. SAT2 mRNA levels increased after amino acid deprivation but not after hyperosmotic shock. Inhibition of p38 activity or transfection with a dominant negative p38 did not alter the response to amino acid starvation but partially blocked the hypertonicity response. Inhibition of the ERK pathway resulted in full inhibition of the adaptive response of system A and no increase in SAT2 mRNA levels, without modifying the response to hyperosmolarity. Similar results were obtained after transfection with a dominant negative JNK1. The CDK2 inhibitor peptide-II decreased the osmotic response in a dose-dependent manner but did not have any effect on the adaptive response of system A. In summary, the previously proposed model of up-regulation of system A after hypertonic shock or after amino acid starvation by separate mechanisms is now confirmed and the two signal transduction pathways have been identified. The involvement of a CDK-cyclin complex in the osmotic response of system A links the activity of this transporter to the increase in cell volume previous to the entry in a new cell division cycle.     

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.     

Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications

Filamentous inclusions of alpha-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes alpha-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant alpha-synuclein proteins. alpha-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr --> Phe alpha-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant alpha-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable alpha-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of alpha-synuclein protein modifications that can influence the formation of stable alpha-synuclein fibrils.     

Field evaluation of efficacy and persistence of an insect vacuum device against the tarnished plant bug (Hemiptera: Miridae) in a day-neutral strawberry field

The efficacy of a vacuuming device to reduce Lygus lineolaris (Palisot de Beauvois) populations was evaluated over 3 yr in a day-neutral strawberry field. Immediately after treatments, a significant reduction of population estimates (by tapping of flower clusters) was observed for adults (75% of the time) and nymphs (50% of the time). Under the same conditions, control (i.e., vacuum turbine off) treatments significantly reduced adult population estimates 25% of the time. Lygus lineolaris tapping samples taken from strawberry plants adjacent to the treated zone did not show significant variations over sampling time, suggesting that no escape behavior occurred. This was supported by adult catches on sticky traps located in zones adjacent to the treated one. Our results suggest that vacuuming had inconsistent effect on tarnished plant bug populations and that L. lineolaris mobility plays a marginal role in the use of this control method.