Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

Accelerated leaf senescence takes part in enhanced resistance in cucumber mosaic virus inoculated pepper leaves

Altered photosynthetic reactions in cucumber mosaic virus (CMV) inoculated leaves of virus resistant lines L113 and L57 and susceptible pepper (Capsicum annuum L.) plants cv. Albena grown in controlled environment and in the field were investigated. The CMV inoculated leaves of virus resistant lines developed different symptoms—necrotic local lesions on L113 and chlorotic spots on L57 while the same leaves of susceptible cv. Albena were symptomless. The changes in Photosystem II (PSII) and PSI electron transport were evaluated by chlorophyll fluorescence, and far-red (FR) light induced leaf absorbance A _810–860. CMV infection caused a decrease in maximal PSII quantum yield, F _v/F _m, in susceptible leaves. Increased non-photochemical fluorescence quenching in CMV-inoculated leaves of both resistant lines were observed. In CMV-inoculated leaves of all tested plants FR light induced P700 oxidation was decreased. In the present study, the viral-infected pepper plants grown in controlled environment to avoid the effects of abiotic factors were used as model system that allow us to investigate the differences in leaf senescence in CMV-inoculated leaves of susceptible and resistant pepper lines expressing different symptoms. Earlier leaf falls of inoculated leaves as a result of accelerated leaf senescence is important for building successful secondary virus resistance strategy following fast responses such as hypersensitive reaction.     

Application of accelerated storage test to lyophilized plant viruses

Summary The preservation of nine plant virus strains of tobamovirus and cucumovirus groups after freeze-drying in different lyophilic forms was examined. Quantitative studies on survival were performed. In tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), accelerated storage test at 70 – 100°C was applied for screening 20 protecting media. A perspective medium, 5% sorbitol, 3.6% dextran, for plant viruses lyophilization with high cryo- and xeroprotective effects was found.     

Differential expression of β-glucosidase in tomato — stress stimuli systems

In the present work the responses of β-glucosidase in leaves of tomato plants subjected to various stress factors of both pathogenic (fungi, bacteria, viruses) and abiotic origin (heat shock) were studied. Biochemical and cytochemical methods were applied. It was established that an increase of β-glucosidase activity is induced uniquely by fungal pathogens. The cytochemical tests confirm the finding. Hence, the conclusion can be drawn that β-glucosidase response is a specific character of fungal pathogenesis in tomato; probably, the enzyme is involved in plant — fungi recognition. The data are in accordance with our previous results on tobacco and wheat — stress stimuli systems.     

Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway

It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Choanocyte ultrastructure in Halisarca dujardini (Demospongiae,Halisarcida)

Understanding poriferan choanocyte ultrastructure is crucial if we are to unravel the steps of a putative evolutionary transition between choanoflagellate protists and early metazoans. Surprisingly, some aspects of choanocyte cytology still remain little investigated. This study of choanocyte ultrastructure in the halisarcid demosponge Halisarca dujardini revealed a combination of minor and major distinctive traits, some of them unknown in Porifera so far. Most significant features were 1) an asymmetrical periflagellar sleeve, 2) a battery of specialized intercellular junctions at the lateral cell surface complemented with an array of lateral interdigitations between adjacent choanocytes that provides a particular sealing system of the choanoderm, and 3) a unique, unexpectedly complex, basal apparatus. The basal apparatus consists of a basal body provided with a small basal foot and an intricate transverse skeleton of microtubules. An accessory centriole, which is not perpendicular to the basal body, is about 45°. In addition, a system of short striated rootlets (periodicity = 50–60 nm) arises from the proximal edge of the basal body and runs longitudinally to contact the nuclear apex. This is the first flagellar rootlet system ever found in a choanocyte. The accessory centriole, the rootlet system, and the nuclear apex are all encircled by a large Golgi apparatus, adding another distinctive feature to the choanocyte cytology. The set of distinct features discovered in the choanocyte of H. dujardini indicates that the ultrastructure of the poriferan choanocyte may vary substantially between sponge groups. It is necessary to improve understanding of such variation, as the cytological features of choanocytes are often coded as characters both for formulation of hypotheses on the origin of animals and inference of phylogenetic relationships at the base of the metazoan tree. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

A novel pathway of ceramide metabolism in Saccharomyces cerevisiae

The hydrolysis of ceramides in yeast is catalysed by the alkaline ceramidases Ypc1p and Ydc1p, two highly homologous membrane proteins localized to the ER (endoplasmic reticulum). As observed with many enzymes, Ypc1p can also catalyse the reverse reaction, i.e. condense a non-esterified fatty acid with PHS (phytosphingosine) or DHS (dihydrosphingosine) and thus synthesize ceramides. When incubating microsomes with [3H]palmitate and PHS, we not only obtained the ceramide PHS-[3H]C16:0, but also a more hydrophobic compound, which was transformed into PHS-[3H]C16:0 upon mild base treatment. The biosynthesis of a lipid with similar characteristics could also be observed in living cells labelled with [14C]serine. Its biosynthesis was dependent on the diacylglycerol acyltransfereases Lro1p and Dga1p, suggesting that it consists of an acylceramide. The synthesis of acylceramide could also be monitored using fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazole)-ceramides as an acceptor substrate for microsomal assays. The Lro1p-dependent transfer of oleic acid on to NBD-ceramide was confirmed by high-resolution Fourier transform and tandem MS. Immunopurified Lro1p was equally able to acylate NBD-ceramide. Lro1p acylates NBD-ceramide by attaching a fatty acid to the hydroxy group on the first carbon atom of the long-chain base. Acylceramides are mobilized when cells are diluted into fresh medium in the presence of cerulenin, an inhibitor of fatty acid biosynthesis.     

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4 Å resolution). hMDD exhibits V_max = 6.1 ± 0.5 U/mg; K_m for ATP is 0.69 ± 0.07 mM and K_m for (R,S) mevalonate diphosphate is 28.9 ± 3.3 μM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a ∼1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K_i = 2.3 ± 0.3 uM) and 6-fluoromevalonate 5-diphosphate (K_i = 62 ± 5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V_max = 0.25 ± 0.02 U/mg and a 15-fold inflation in K_m for mevalonate diphosphate. N17A’s K_i values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8 Å) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.