Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR

Low-affinity state beta1-adrenoceptor (beta1-AR) was functionally expressed in some blood vessels and was different from beta1, beta2 and beta3-AR. In rat aorta, low-affinity state beta1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin-angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration-response curves to low-affinity state beta1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state beta1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state beta1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K(ATP)+ channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state beta1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.     

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.     

Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze–thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress—including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.Human serum albumin contains a single free cysteine residue (Cys34) that is susceptible to oxidation via disulfide-bond formation with free cysteine amino acids, resulting in S-cysteinylated (oxidized) albumin (1). Human apolipoprotein A-I (apoA-I)¹ contains three methionine residues (Met86, Met112, and Met148) that can be oxidized to sulfoxides (2–4). The oxidized forms of both of these plasma/serum (P/S) proteins have been proposed as markers of conditions involving oxidative stress (5–9), including atherosclerosis (6–8). These proteins are readily analyzed intact via mass spectrometry in a single run using simple dilute-and-shoot techniques; thus if scientifically suitable, they are well positioned analytically to serve as clinical markers. At least some evidence exists, however, that both albumin (10) and apoA-I (6) are susceptible to artifactual oxidation ex vivo. Notably, the scientific literature in recent years has been relatively quiet with regard to both of these markers. We suspected that spontaneous artifactual oxidation of these proteins ex vivo led to their initial implication as markers of disease, but that the same phenomenon might have confounded efforts to clinically validate them (11, 12). Thus we undertook systematic studies of albumin and apoA-I oxidation ex vivo and found evidence indicating that rather than serving as markers of disease, oxidized albumin and apoA-I may serve as markers for improper handling and storage of blood P/S.Improper biospecimen handling and storage can contribute to sample measurements that do not accurately reflect biological reality in vivo (13–16). This may introduce bias in analytical results, limiting the capacity for meaningful comparisons among patient groups (17–19). Thus careful pre-analytical sample handling is a vital component of both clinical investigation and biomarker research. For clinical assays, parameters that define proper sample handling and storage are generally determined during assay validation and are typically incorporated into laboratory standard operating procedures. In blood P/S-based biomarker development work, however, verification of sample integrity is sometimes overlooked or considered only as an afterthought. Contributing to this phenomenon is that fact that there are no universally accepted, globally applicable endogenous reference markers of P/S integrity. Indeed, there likely does not exist a single, individual marker capable of meeting this broad specification. Nonetheless, identification and standardization of quality control markers that cover this specific scope of application (i.e. proper storage conditions for blood P/S) represent an important goal of biobanking-related research (16, 20).Betsou et al. (16) recently outlined and ranked some of the strongest candidates for use as quality control tools in biomarker research. Within the scope of tools for assessing proper handling and storage of P/S samples, nearly all markers are founded on the quantification of a nominal protein via a molecular-recognition-based assay. As a result, the indication of a loss of specimen integrity lies in an apparent loss of the target protein beyond the normal human reference range. Such loss is often ascribed to “degradation” and in many cases likely happens because of residual proteolytic activity that occurs at temperatures above the sample freezing point. In other cases, loss of the protein marker may be due to misfolding caused by repeated freeze–thaw cycles.Though not frequently discussed, protein “degradation” ex vivo may also have roots in oxidative processes that are capable of disrupting protein–antibody interactions that serve as the basis for protein quantification. It is well known that in the absence of special precautions, disulfide bonds will form spontaneously between cysteine thiols. We have previously shown that this requires only the presence of atmospheric oxygen and trace metals and proceeds through a cysteine sulfenic acid intermediate (21). This mechanism also applies to S-cysteinylation of albumin (22–24), though disulfide exchange with cystine may be operative in P/S as well. Likewise, it is known that methionine-containing proteins and peptides will oxidize to sulfoxides spontaneously in the presence of atmospheric oxygen (25, 26); indeed, artifactual sulfoxidation of methionine residues in peptide-based proteomics work is well known. Oxidative modifications such as these have the potential to disrupt antibody interactions with the oxidized protein, resulting in low readings in molecular-recognition-based assays. Thus protein oxidation merits investigation as a protein “degradation” pathway.As pointed out by Betsou et al. (16), markers that are highly sensitive to variations in specimen storage and handling conditions are likely to be the most useful. A considerable degree of change that occurs rapidly under undesirable conditions to which samples may be exposed, such as the state of being incompletely frozen (which for blood plasma occurs at temperatures above −30 °C (27–30)), is something to be sought after in a biospecimen-integrity marker. Herein we describe simple methods for the simultaneous relative quantification of oxidized albumin and apoA-I and present evidence that albumin and apoA-I can undergo major increases in oxidation ex vivo and thus may be useful markers of P/S specimen integrity.     

Anopheles gambiae innate immunity

The successful development of Plasmodium in Anopheles mosquitoes is governed by complex molecular and cellular interactions that we are just beginning to understand. Anopheles immune system has received particular attention as genetic evidence points clearly to its critical role in eliminating the majority of parasites invading the midgut epithelium. Several factors regulating Plasmodium development have been identified and tentatively assigned to the individual steps leading to mosquito immune reactions; non-self-recognition, signal modulation, signal transduction and effector mechanisms. Detailed knowledge of these steps and their underlying molecular mechanisms may offer novel perspectives to abort Plasmodium development in the vector. Here, we summarize our current knowledge of mosquito innate immunity highlighting both, recent advances and areas where additional research is required.     

Role of the juxtamembrane region of cytoplasmic loop 3 in the gating and conductance of the cystic fibrosis transmembrane conductance regulator chloride channel

Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl(-) conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl(-) permeation pathway to exert any influence on permeating Cl(-) ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl(-) ions use other routes to access the transmembrane pore.     

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 μM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 μM acetamiprid and imidacloprid had a strong effect. 1 and 10 μM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.     

Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study

Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.     

Characterisation of Pellicles Formed by Acinetobacter baumannii at the Air-Liquid Interface

The clinical importance of Acinetobacter baumannii is partly due to its natural ability to survive in the hospital environment. This persistence may be explained by its capacity to form biofilms and, interestingly, A. baumannii can form pellicles at the air-liquid interface more readily than other less pathogenic Acinetobacter species. Pellicles from twenty-six strains were morphologically classified into three groups: I) egg-shaped (27%); II) ball-shaped (50%); and III) irregular pellicles (23%). One strain representative of each group was further analysed by Brewster’s Angle Microscopy to follow pellicle development, demonstrating that their formation did not require anchoring to a solid surface. Total carbohydrate analysis of the matrix showed three main components: Glucose, GlcNAc and Kdo. Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation. Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091. The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.