Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: morphology, nuclear and chloroplast DNA data

Parsimony analyses of morphology, restriction sites of the cpDNA, sequences from the nuclear, ribosomal internal transcribed spacer (ITS), and the chloroplast gene rbcL were performed to asses tribal and generic relationships in the subfamily Ixoroideae (Rubiaceae). The tribes Vanguerieae and Alberteae (Antirheoideae) are clearly part of Ixoroideae, as are some Cinchonoideae taxa. Pavetteae should exclude Ixora and allies, which should be recognized as the tribe Ixoreae. Heinsenia, representing Aulacocalyceae, is part of Gardenieae, as is Duperrea, a genus earlier placed in Pavetteae. Posoqueria and Bertiera and the taxa in the subtribe Diplosporinae should be excluded from Gardenieae. Bertiera and three Diplosporinae taxa are part of Coffeeae, while Cremaspora (Diplosporinae) is best housed in a tribe of its own, Cremasporeae. The mangrove genus Scyphiphora, recently placed in Diplosporinae, is closer to Ixoreae and tentatively included there. The combined analysis resulted in higher resolution compared to the separate analyses, exemplifying that combined analyses can remedy the incapability of one data set to resolve portions of a phylogeny. Twenty-four new rbcL sequences representing all five Ixoroideae tribes (sensu Robbrecht) are presented.     

Induced Levels of Heat Shock Proteins in a dnaK Mutant of Lactococcus lactis

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaKΔ2 mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (dnaKΔ1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L. lactis. For Bacillus subtilis, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579–4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins. The present data from L. lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.     

The influence of aquatic surface respiration (ASR) on cardio-respiratory function of the serrasalmid fish Piaractus mesopotamicus

When exposed to severely hypoxic water, many teleosts skim the better oxygenated surface layer (aquatic surface respiration, ASR). Information is scarce concerning the thresholds triggering ASR and its cardio-respiratory consequences. To assess the ambient conditions leading to ASR and to evaluate its effects on cardio-respiratory function, we exposed specimens of Piaractus mesopotamicus to gradual hypoxia (water oxygen tension ranging from 120 to 10 torr) with or, alternatively, without access to the surface. Concurrently, ASR, cardiac and respiratory frequencies, O₂ uptake and gill ventilation were monitored. With surface access, ASR developed below the critical tension for O₂ uptake (34 torr) by normal gill ventilation. Moreover, the time spent in ASR increased with prolonged hypoxic exposure to a maximum of 95% of total time. Without surface access, the species exhibited hypoxic bradycardia, that had not occurred in the group with fully developed ASR. Even without ASR, P. mesopotamicus recovered readily from hypoxic exposure, showing that this species possesses a number of mechanisms to cope with environmental hypoxia.     

First Administration of the Fc-Attenuated Anti-β Amyloid Antibody GSK933776 to Patients with Mild Alzheimer’s Disease: A Randomized,Placebo-Controlled Study

Objective

To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-β amyloid (Aβ) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer’s disease (AD) or mild cognitive impairment (MCI).

Methods

This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001–6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1–6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436).

Results

There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or –hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t_1/2) was 10–15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aβ42 and Aβ increased whereas plasma levels of free Aβ decreased dose dependently; no changes were observed for placebo. For total Aβ42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aβ the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aβ showed increases from baseline to week 12 for Aβ X–38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aβ X–42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses.

Conclusion

In this FTIH study the Fc-inactivated anti-Aβ mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI.

Trial Registration

ClinicalTrials.gov NCT00459550     

Column-switching HPLC–MS/MS analysis of ropivacaine in serum,ultrafiltrate and drainage blood for validating the safety of blood reinfusion

A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) method, using back-flush column-switching was developed for total drug concentrations of ropivacaine in serum and drainage blood in the measuring range 0.1–10 μg/mL. Samples were diluted with internal standard (²H₇-ropivacaine) and extraction buffer, centrifuged and injected directly onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, ropivacaine was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electro spray ionisation and multiple reaction monitoring (MRM) of the transitions m/z 275 → m/z 126 and m/z 282 → m/z 133 for ropivacaine and ²H₇-ropivacaine, respectively. Accuracy (bias-%) was −1.5 to 5.8% and intermediate precision (C.V.) was 1.4–3.1%. The low sample amount required (10 μL), high specificity and short run time (6 min) makes it very suitable for determination of ropivacaine. Using the same methodology as described above and 200 μL ultrafiltrate, the free drug concentrations of ropivacaine in serum could be precisely determined with a C.V. below 3%. The method was used to investigate the safety of reinfusion of drainage blood after knee and hip arthroplasty when ropivacaine (Naropin^®) was used for local analgesia. Data for 30 patients are summarised.     

Heparin promotes fibrillation of most phenol-soluble modulin virulence peptides from Staphylococcus aureus

Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.     

Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.