Evidence of hemispheric specialization in marmosets (Callithrix penicillata) using tympanic membrane thermometry

Recent studies have employed tympanic thermometry to assess lateralization of cognitive and emotional functions in primates. However, no studies using this technique have investigated the possibility of hemispheric specialization in New World monkeys. Therefore, the aim of the present study was to investigate tympanic membrane (TM) temperature asymmetries and their possible correlation with stress responses in marmosets (Callithrix penicillata). Infrared TM thermometry was completed bilaterally in 24 animals (14 males and 10 females) during a stressful situation of capture and restraint. There were no significant differences between gender. A significant negative correlation was observed between TM temperature of the right ear and the number of captures (r = -0.633; P<0.001). Subjects with a more frequent previous history of captures (5 to 9 captures; N = 11) showed lower TM temperature when compared to those with fewer previous captures (1 to 4 captures; N = 13). No differences were observed for the left TM temperature. These results suggest that under intense emotional challenge (capture and restraint) there is a stronger activation of the neural structures situated in the right brain hemisphere. Taken together, the data reveal for the first time evidence of hemispheric specialization in emotional physiological processing in a New World monkey.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

The effect of hydrostatic pressure on membrane-bound proteins

Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.

Unraveling the functional divergence of membrane-bound pyrophosphatases

Inorganic pyrophosphatases (PPases) are enzymes that hydrolyze pyrophosphate (PPi)which is produced as a byproduct in many important growth related processes e.g. in the biosynthesis of DNA, proteins and lipids. PPases can be either soluble or membranebound. Membrane-bound PPases (mPPases) are ion transporters that couple the energy released during PPi hydrolysis to Na+ or H+ transport. When I started the project, only three Na+-transporting mPPases were known to exist. In this study, I aimed to confirm if Na+-transport is a common function of mPPases. Furthermore, the amino acid residues responsible for determining the transporter specificity were unknown. I constructed a phylogenetic tree for mPPases and selected the representative bacterial and archaeal mPPases to be investigated. I expressed different prokaryotic mPPases in Escherichia coli, isolated these as inverted membrane vesicles and characterized their functions. In the first project I identified four new Na+-PPases, two K+-dependent H+-PPases and one K+-independent mPPase. The residues determining the transporter specificity were identified by site-directed mutagenesis. I showed that the conserved glutamate residues are important for specificity, though are not the only residues that influence it. This research clarified the ion transport specificities throughout the mPPase phylogenetic tree, and revealed that Na+ transport is a widespread function of mPPases. In addition, it became clear that the transporter specificity can be predicted from the amino acid sequence in combination with a phylogenetic analysis. In the second project, I identified a novel class of mPPases, which is capable of transporting both Na+ and H+ ions and is mainly found in bacteria of the human gastrointestinal tract. The physiological role of these novel enzymes may be to help the bacteria survive in the demanding conditions of the host. In the third project, I characterized the Chlorobium limicola Na+-PPase and found that this and related mPPases are able to transport H+ ions at subphysiological Na+ concentrations. In addition, the H+-transport activity was shown to be a common function of all studied Na+-PPases at low Na+ concentrations. I observed that mutating gate-lysine to asparagine eliminated the H+ but not the Na+ ion transport function, indicating the important role of the residue in the transport of H+. In the fourth project, I characterized the unknown and evolutionary divergent mPPase clade of the phylogenetic tree. The enzymes belonging to this clade are able to transport H+ ions and, based on their sequence, were expected to be K+- and Na+-independent. The sequences of membrane-bound PPase are usually highly conserved, but the enzymes belonging to this clade are more divergent and usually contain 100−150 extra amino acid residues compared to other known mPPases. Despite the vast sequence differences, these mPPases have the full set of important residues and, surprisingly, are regulated by Na+ and K+ ions. These enzymes are mainly of bacterial origin.

Leishmania (Leishmania) chagasi-infected mice as a model for the study of glomerular lesions in visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Glomerular filtration is reduced by high tidal volume ventilation in an in vivo healthy rat model

Mechanical ventilation has been associated with organ failure in patients with acute respiratory distress syndrome. The present study examines the effects of tidal volume (V T) on renal function using two V T values (8 and 27 mL/kg) in anesthetized, paralyzed and mechanically ventilated male Wistar rats. Animals were randomized into two groups of 6 rats each: V T8 (V T, 8 mL/kg; 61.50 ± 0.92 breaths/min; positive end-expiratory pressure, 3.0 cmH2O; peak airway pressure (PAW), 11.8 ± 2.0 cmH2O), and V T27 (V T, 27 mL/kg; 33.60 ± 1.56 breaths/min; positive end-expiratory pressure, none, and PAW, 22.7 ± 4.0 cmH2O). Throughout the experiment, mean PAW remained comparable between the two groups (6.33 ± 0.21 vs 6.50 ± 0.22 cmH2O). For rats in the V T27 group, inulin clearance (mL·min-1·body weight-1) decreased acutely after 60 min of mechanical ventilation and even more significantly after 90 min, compared with baseline values (0.60 ± 0.05 and 0.45 ± 0.05 vs 0.95 ± 0.07; P < 0.001), although there were no differences between groups in mean arterial pressure or gasometric variables. In the V T8 group, inulin clearance at 120 min of mechanical ventilation remained unchanged in relation to baseline values (0.72 ± 0.03 vs 0.80 ± 0.05). The V T8 and V T27 groups did not differ in terms of serum thiobarbituric acid reactive substances (3.97 ± 0.27 vs 4.02 ± 0.45 nmol/mL) or endothelial nitric oxide synthase expression (94.25 ± 2.75 vs 96.25 ± 2.39%). Our results show that glomerular filtration is acutely affected by high tidal volume ventilation but do not provide information about the mechanism.

Monitoring renal function: measured and estimated glomerular filtration rates - a review

Chronic kidney disease (CKD) is a world-wide public health problem, with adverse outcomes of kidney failure, cardiovascular disease, and premature death. This finding has led to the hypothesis that earlier recognition of kidney disease and successful intervention may improve outcome. The National Kidney Foundation, through its Kidney Disease Outcomes Quality Initiative (K/DOQI), and other National institutions recommend glomerular filtration rate (GFR) for the definition, classification, screening, and monitoring of CKD. Blood creatinine clearance, the most widely used clinical marker of kidney function, is now recognized as an unreliable measure of GFR because serum creatinine is affected by age, weight, muscle mass, race, various medications, and extra-glomerular elimination. Cystatin C concentration is a new and promising marker for kidney dysfunction in both native and transplanted kidneys. Because of its low molecular weight, cystatin C is freely filtered at the glomerulus and is almost completely reabsorbed and catabolized, but not secreted, by tubular cells. Given these characteristics, cystatin C concentration may be superior to creatinine concentration in detecting chronic kidney disease. This review aims to evaluate from recent literature the clinical efficiency and relevance of these GFR markers in terms of screening CKD.

Glomerular damage as a predictor of renal allograft loss

Interstitial fibrosis and tubular atrophy (IF/TA) are the most common cause of renal graft failure. Chronic transplant glomerulopathy (CTG) is present in approximately 1.5-3.0% of all renal grafts. We retrospectively studied the contribution of CTG and recurrent post-transplant glomerulopathies (RGN) to graft loss. We analyzed 123 patients with chronic renal allograft dysfunction and divided them into three groups: CTG (N = 37), RGN (N = 21), and IF/TA (N = 65). Demographic data were analyzed and the variables related to graft function identified by statistical methods. CTG had a significantly lower allograft survival than IF/TA. In a multivariate analysis, protective factors for allograft outcomes were: use of angiotensin-converting enzyme inhibitor (ACEI; hazard ratio (HR) = 0.12, P = 0.001), mycophenolate mofetil (MMF; HR = 0.17, P = 0.026), hepatitis C virus (HR = 7.29, P = 0.003), delayed graft function (HR = 5.32, P = 0.016), serum creatinine ≥1.5 mg/dL at the 1st year post-transplant (HR = 0.20, P = 0.011), and proteinuria ≥0.5 g/24 h at the 1st year post-transplant (HR = 0.14, P = 0.004). The presence of glomerular damage is a risk factor for allograft loss (HR = 4.55, P = 0.015). The presence of some degree of chronic glomerular damage in addition to the diagnosis of IF/TA was the most important risk factor associated with allograft loss since it could indicate chronic active antibody-mediated rejection. ACEI and MMF were associated with better outcomes, indicating that they might improve graft survival.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Frequency of primary glomerular disease in northeastern China

Most frequently reported Chinese renal biopsy data have originated from southeastern China. The present study analyzed the renal biopsy data from northeastern China. The records of 1550 consecutive native patients who were diagnosed with primary glomerular diseases (PGD) after renal biopsy at our hospital during 2005-2009 were used. These patients were divided into four age groups for stratified analysis: <15, 15-44, 45-59, and ≥60 years old. Among PGD, minimal change disease (MCD) was the most common histologically diagnosed disease (30.7%), followed by IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis (MsPGN), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN), focal segmental glomerulosclerosis (FSGS), and endocapillary proliferative glomerulonephritis (EnPGN). MCD was the disease most frequently observed (43.7%) in the <15-year-old group. MsPGN was the most common disease in the elderly group (38.1%). MsPGN was more prevalent in females (27.8%), whereas MCD was more prevalent in males (35.3%). Primary glomerular diseases constituted the most commonly encountered group of diseases with a high prevalence of MCD, which predominantly affected males and young adults. The prevalence of MCD was high in northeastern China. Further study is necessary to expand the epidemiologic data available for renal disease in China.