Effects of 7-day amino acid infusion on renal growth, function, and renin-angiotensin system in fetal sheep

These experiments examined whether renal growth and the fetal renin-angiotensin system could be stimulated by infusion of amino acids and whether chronic amino acid infusions restored glomerulotubular balance, which had been disrupted during 4-h infusions. Five fetal sheep aged 122 +/- 1 days gestation received an infusion of alanine, glycine, proline and serine in 0.15 M saline at 0.22 mmol/min for 7 days. Six control fetuses were given saline at the same rate (5 ml/h). Kidney wet weights after amino acid infusion were 28% larger than control fetuses (P < 0.05), and renal angiotensinogen mRNA levels were approximately 2.6-fold higher (P < 0.005). Circulating renin levels and renal renin mRNA levels were suppressed (P < 0.05), and renal renin protein levels tended to be lower. Arterial pressure was increased, and there was a marked, sustained natriuresis and diuresis. Glomerular filtration rate and filtered sodium were approximately two-fold higher throughout infusion (P < 0.05). Fractional proximal sodium reabsorption, suppressed at 4 h (from 73.4 +/- 6.5 to 53.7 +/- 10.2%), did not return to control levels (36.1 +/- 3.4% on day 7, P < 0.05). Distal sodium reabsorption was markedly increased (from 79 +/- 25 to 261 +/- 75 mumol/min by day 7, P < 0.005), but this was not sufficient to restore glomerulotubular balance. The resultant high rates of sodium excretion led to hyponatremia and polyhydramnios. In conclusion, long-term amino acid infusions increased renal angiotensinogen gene expression, kidney weight, and distal nephron sodium reabsorptive capacity but failed to restore proximal and total glomerulotubular balance.     

The spore‐associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild‐type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID₅₀) of spores was significantly higher (2‐logs) in the bclA1^? mutant compared to the isogenic wild‐type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild‐type spores. bclA1^? spores germinated faster than wild‐type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre‐spore germination) stages of infection. We also show that the ID₅₀ was higher in mice infected with R20291, a ‘hypervirulent’ 027 strain, that carries a truncated BclA1 protein.     

Phosphatidic acid potentiates Gαq stimulation of phospholipase C-β1 signaling

Phosphatidic acid (PA) is interactive with Gα_q-linked agonists to stimulate GPCR signaling via phospholipase C-β₁ (PLC-β₁). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β₁ activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β₁ and Gα_q. Activated Gα_q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β₁ activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β₁ implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β₁ to both PMA and carbachol. PA alone thus has little effect on PLC-β₁ activity, but PA and PLD1 synergize with activated Gα_q to stimulate PLC-β₁ signaling. Coordinate interaction with activated Gα_q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.     

Potential advantages of acute kidney injury management by mesenchymal stem cells

Mesenchymal stem cells are currently considered as a promising tool for therapeutic application in acute kidney injury (AKI) management. AKI is characterized by acute tubular injury with rapid loss of renal function. After AKI, inflammation, oxidative stress and excessive deposition of extracellular matrix are the molecular events that ultimately cause the end-stage renal disease. Despite numerous improvement of supportive therapy, the mortality and morbidity among patients remain high. Therefore, exploring novel therapeutic options to treat AKI is mandatory. Numerous evidence in animal models has demonstrated the capability of mesenchymal stem cells (MSCs) to restore kidney function after induced kidney injury. After infusion, MSCs engraft in the injured tissue and release soluble factors and microvesicles that promote cell survival and tissue repairing. Indeed, the main mechanism of action of MSCs in tissue regeneration is the paracrine/endocrine secretion of bioactive molecules. MSCs can be isolated from several tissues, including bone marrow, adipose tissue, and blood cord; pre-treatment procedures to improve MSCs homing and their paracrine function have been also described. This review will focus on the application of cell therapy in AKI and it will summarize preclinical studies in animal models and clinical trials currently ongoing about the use of mesenchymal stem cells after AKI.     

Characterization of bovine serum albumin glycated with glucose, galactose and lactose

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.     

Different responses in the expression of alginases,alginate polymerase and acetylation genes during alginate production by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> under oxygen-controlled conditions

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate \((q_{{{\text{O}}_{ 2} }} )\) from 10.9 to 45.3 mmol g^?1 h^?1 by changes in the dilution rate (D) from 0.06 to 0.10 h^?1, whereas under 1% DOT the \(q_{{{\text{O}}_{ 2} }}\) was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.     

Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin‐embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.     

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.     

T cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies

Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect. In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly, 7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic sites. More strikingly, depletion of CD8⁺ and CD4⁺ T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant. This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction of an adjuvant effect. This work was supported by the Cuban Government.