Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.     

Heat resistance studies of yeasts; vegetative cells versus ascospores: erythromycin inhibition of sporulation in Kluyveromyces and Saccharomyces species

A comparative study of the heat resistance ( D ₆₀ values) of four Saccharomyces spp. and two Kluyveromyces spp. (21 strains) showed a 30–350-fold higher heat resistance of ascospores than of vegetative cells. It was also observed that small numbers of ascospores exhibiting a considerably higher heat resistance can easily be formed, even in a complete vegetative growth medium. This phenomenon may have led most other authors to report none or only slight differences between the heat resistance of yeast ascospores and their vegetative cells. Until more information has been collected about the ascospore load of acid (fruit) products and their heat resistance, accurate calculations of the minimum F values for heat preservation of these products may not be possible.     

Anti-cholesterol antibody levels in hereditary angioedema

Hereditary angioedema (HAE) is a rare disorder caused by the deficiency of the C1-inhibitor gene (C1INH) and characterized by recurrent bouts of angioedema. Autoimmune disorders frequently occur in HAE. Previously we found, that danazol has an adverse effect on serum lipid profile: reduced high-density lipoprotein (HDL) and elevated low-density lipoprotein (LDL) cholesterol levels are associated with long-term prophylactic use, whereas total cholesterol levels are unchanged. Our aim was to study the anti-cholesterol antibody (ACHA) production in HAE patients and compare it with those of healthy blood donors, and to investigate the possible associations between ACHA levels and serum lipid profile alterations caused by danazol. Anti-cholesterol IgG levels were measured by ELISA and their correlation with serum concentrations of total cholesterol, HDL, LDL, triglycerides was determined in HAE patients receiving/not receiving danazol. Serum ACHA levels were significantly higher in HAE patients, compared to healthy blood donors (P<0.0001). Longterm danazol prophylaxis had no effect on serum ACHA levels in HAE patients. However, we found a significant, negative correlation between ACHA levels and serum total cholesterol (r=-0.4033, P=0.0200), LDL (r=-0.4565, P=0.0076) and triglyceride (r=-0.4230, P=0.0121) levels only in danazol-treated patients, but not in HAE patients who did not receive long-term prophylaxis. Patients with HAE have higher baseline ACHA levels compared to healthy subjects, and this might reflect polyclonal B-cell activation. The latter would be a potential explanation for the lack of an increased incidence of infectious diseases in HAE patients, but might lead to increased autoimmunity.     

Autophagy-related lipase FgATG15 of Fusarium graminearum is important for lipid turnover and plant infection

Autophagy is a non-selective degradation pathway in eukaryotic cells that is conserved from yeasts to humans. Autophagy is involved in the virulence of several pathogenic fungi such as Magnaporthe grisea or Colletotrichum orbiculare. In the current study, we identified and disrupted an autophagy-like lipase FgATG15 in Fusarium graminearum. We showed that FgATG15 exhibits lipase activity when heterologously expressed in P. pastoris. We used a gene deletion approach to characterize the function of the enzyme. We demonstrate that FgATG15 is involved in fungal growth and aerial hyphae production. FgATG15 is also involved in conidia production and germination, and disruption of FgATG15 led to aberrant conidia shapes. FgATG15 disruptants were reduced in storage lipid degradation under starvation conditions, implicating FgATG15's involvement in lipid turnover. Moreover, wheat head infection by the disruptants was severely attenuated, indicating the involvement of FgATG15 in pathogenesis. Additionally, we found that the deoxynivalenol levels of FgATG15 disruptants were significantly decreased compared with the wild type strain. Taken together, we show that FgATG15 is involved in numerous developmental processes and could be exploited as an antifungal target.     

Dopamine and alcohol relapse: D1 and D2 antagonists increase relapse rates in animal studies and in clinical trials

A considerable number of animal studies on the effects of dopaminergic agents on alcohol intake behavior have been performed. Acute alcohol administration in rats induces dopamine release in the caudate nucleus and in the nucleus accumbens, an effect related among others to reinforcement. It has been repeatedly suggested that D1 and D2 receptor activation mediates reward. As alcohol consumption and dopaminergic transmission seem to have a close relationship, all kinds of dopaminergic agents may be regarded as putative therapeutics for preventing relapse. In a prospective European double-blind multicenter clinical trial, comparing the D1, D2, D3 antagonist flupenthixol and placebo in 281 chronic alcohol-dependent patients (27.4% women), the application of the Lesch typology made an outcome differentiation possible. It could be shown in which patients flupenthixol administration was followed by a significantly higher relapse rate and in which patient groups no differences were found when compared to placebo.     

Keeping the intracellular vitamin C at a physiologically relevant level in endothelial cell culture

It is generally accepted that the addition of vitamin C to cell culture medium improves cell growth. However, once added, the vitamin C concentration declines rapidly. This situation differs from the in vivo environment where the endothelium is constantly supplied with ascorbate from the blood. With a focus on intracellular vitamin C, we simulated constant supply of ascorbate by the hourly addition of freshly prepared medium containing 75 μM ascorbate and subsequently compared it with more practical regimens using combinations of ascorbate and 2-phosphoascorbate. We found that a single supplement of ascorbate and 2-phosphoascorbate adequately maintains intracellular vitamin C at physiological levels for up to 72 h.     

Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise

During prolonged, low intensity exercise, the type of substrate utilized varies with time. If 5' AMP-activated protein kinase (AMPK) regulates muscle metabolism during exercise, signaling through AMPK would be expected to change in concordance with changes in substrate utilization. Six healthy, young males cycled (approximately 45% VO(2peak)) until exhaustion (approximately 3.5h). During exercise, leg glucose uptake and rate of glycogenolysis gradually decreased whereas free fatty acid uptake gradually increased. In the thigh muscle, the alpha AMPK subunits became progressively more phosphorylated on Thr(172) during exercise eliciting a parallel increase in alpha2 but not alpha1 AMPK activity. In contrast, after 1h of exercise, Ser(221) phosphorylation of acetyl-CoA carboxylase-beta (ACCbeta) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of alpha2 AMPK, alpha1 AMPK or ACCbeta did not change with time. These data suggest that AMPK signaling is not a key regulatory system of muscle substrate combustion during prolonged exercise and that marked activation of AMPK via phosphorylation is not sufficient to maintain an elevated ACCbeta Ser(221) phosphorylation during prolonged exercise.     

The remote substrate binding subsite -6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism.

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-, beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had decreased k(cat) values for beta-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite -6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite -6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the alpha-amylase family.     

Phosphoinositide-generated messengers in cardiac signal transduction

A multitude of agonists like e.g. endothelin-I, angiotensin-II, serotonin, thrombin, histamine and vasopressin as well as ₁-adrenergic and muscarinic stimulation lead to stimulation of the phosphoinositide cycle in the heart. Besides this seven membrane spanning-domain receptor-coupled stimulation of the key enzyme of the phosphoinositide cycle, phospholipase C-, another class of hormones, growth factors, also couple to the phosphoinositide cycle, now through receptors with intrinsic tyrosine kinase activity that can phosphorylate and stimulate the phospholipase C- isozyme. In this review we summarize the multitude of receptor (sub)types, G-protein-subunit- and phospholipase C-isozymes that are present in the heart. Furthermore, generation of second messengers and cellular responses are described together with the (patho)physiological implications for the heart of phosphoinositide cycle activation and second messenger accumulation.