The secretome of Pleurotus sapidus

Due to their unique capability to attack lignified biopolymers, extracellular enzymes of white-rot fungi enjoy an increasing interest in various fields of white biotechnology. The edible fungus Pleurotus sapidus was selected as a model organism for the analysis of the secretome by means of 2-DE. For enzyme production, the fungus was grown in submerged cultures either on peanut shells or on glass wool as a carrier material. Identification of the secreted enzymes was performed by tryptic digestion, ESI-MS/MS ab initio sequencing, and homology searches against public databases. The spectrum of secreted enzymes comprised various types of hydrolases and lignolytic enzymes of the manganese peroxidase/versatile peroxidase family. While peptidases were secreted mainly by the cultures grown on peanut shells, versatile peroxidase type enzymes dominated in the cultures grown on glass wool.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Evidence for quantitative trait loci affecting twinning rate in North American Holstein cattle

Twinning in dairy cattle has been associated with many negative health and reproductive events that cause economic loss to the producer. Reports have suggested that twinning rates are increasing and that there may be a positive relationship between milk production and twinning frequency. Putative quantitative trait loci (QTL) for twinning and ovulation rate on bovine chromosomes 5, 7, 19 and 23 have been previously identified in other populations. The objective of this study was to detect and possibly confirm the existence and effects of these QTL in the North American Holstein population. Half-sib families of 20 North American Holstein sires with above average twinning rate predicted transmitting abilities (PTA) comprised the sample population under investigation. Twinning rate PTA values had been estimated from calving data. DNA extracted from semen samples was analysed using 45-61 microsatellite markers across the four chromosomes. Marker heterozygosity of the patriarchs averaged 62%. Evidence of twinning QTL was found in multiple families on chromosomes 5, 7 and 23 and in one family on chromosome 19. Four of the sires formed one three-generation family: one sire and three half-sib sons with sons of their own. This extended family was analysed with additional markers confirming a twinning QTL of significant size on chromosome 5.     

Cardiovascular responses to a high-volume continuous circuit resistance training protocol

The purpose of this investigation was to determine the level of cardiovascular stress elicited by continuous and prolonged circuit resistance training (CRT). Each of the 11 men who volunteered as a subject were tested to determine oxygen consumption and heart rate responses to a submaximal and maximal treadmill protocol and a CRT session consisting of 10 exercises and 10 repetitions at 40% of 1 repetition maximum (1RM) for each station with 4.6 circuits performed. The physiological stress of the CRT in this study was evident by the sustained heart rate of more than 70% of maximum for 16.6 minutes, with the last 12 minutes at more than 80%. Despite the large anaerobic component in CRT, Vo(2) was sustained at 50% or more of maximum for the final 12 minutes. Treadmill running, involving large muscle groups, increased Vo(2) more rapidly than CRT, where alternating larger and smaller muscle groups were used. In addition, at the same Vo(2) heart rate differed significantly between the 2 modes of activity. Heart rate in CRT was higher (at 165) than the heart rate of 150 found during treadmill running at the same 50% Vo(2). Such workouts may be used in a training cycle in classical linear periodization or in a nonlinear program day targeting local muscular endurance under intense cardiorespiratory conditions, which may help individuals develop enhanced toleration of physiological environments where high cardiovascular demands and higher lactate concentrations are present.     

X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism

The neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is caused by ABCD1 mutations and characterized by very long-chain fatty acid (VLCFA) accumulation. Cholesterol-lowering normalized VLCFA in fibroblasts and plasma of X-ALD patients. We show that in cultured cells, cholesterol-loading induces ABCD1. In X-ALD mice, plasma cholesterol is elevated and not further increasable by cholesterol-feeding, whereas hepatic HMG-CoA reductase and Abcd2 are downregulated. Upon cholesterol modulation, brain VLCFA increased in X-ALD mice, but decreased in controls. In murine X-ALD fibroblasts, cholesterol-lowering did not normalize VLCFA. Thus, ALDP-deficiency and VLCFA are linked to cholesterol but species differences complicate evaluating cholesterol-lowering drugs in X-ALD mice.     

Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states

Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically labeled with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) and fluorescence quenching experiments were carried out to determine the accessibility of this probe at Lys-553 in both the strongly and weakly actin-bound states of the MgATPase cycle. Solvent quenchers of varying charge [nitromethane, (2,2,6, 6-tetramethyl-1-piperinyloxy) (TEMPO), iodide (I(-)), and thallium (Tl(+))] were used to assess both the steric and electrostatic accessibilities of the FHS probe at Lys-553. In the strongly bound rigor (nucleotide-free) and MgADP states, actin offered no protection from solvent quenching of FHS by nitromethane, TEMPO, or thallium, but did decrease the Stern-Volmer constant by almost a factor of two when iodide was used as the quencher. The protection from iodide quenching was almost fully reversed with the addition of 150 mM KCl, suggesting this effect is ionic in nature rather than steric. Conversely, actin offered no protection from iodide quenching at low ionic strength during steady-state ATP hydrolysis, even with a significant fraction of the myosin heads bound to actin. Thus, the lower 50 kD subdomain of myosin containing Lys-553 appears to interact differently with actin in the weakly and strongly bound states.     

Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is a member of the ATP-binding cassette transporter family. The most conserved features of this family are the nucleotide-binding domains. As in other members of this family, these domains bind and hydrolyze ATP; in CFTR this opens and closes the channel pore. The recent crystal structures of related bacterial transporters show that an aromatic residue interacts with the adenine ring of ATP to stabilize nucleotide binding. CFTR contains six aromatic residues that are candidates to coordinate the nucleotide base. We mutated each to cysteine and examined the functional consequences. None of the mutations disrupted channel function or the ability to discriminate between ATP, GTP, and CTP. We also applied [2-(triethylammonium)ethyl] methanethiosulfonate to covalently modify the introduced cysteines. The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr(1219) and Phe(446) were not important for nucleotide binding. The results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in some bacterial transporters. Taken together with earlier work, this study points to a model in which most of the binding energy for ATP is contributed by the phosphate groups.     

Studies of the complexation of sugars by diffusion-ordered NMR spectroscopy

In the presence of lanthanide cations, some sugars with a special relationship between their hydroxyl groups are able to form complexes in water. Diffusion-ordered NMR spectroscopy (DOSY) can be used as a tool to distinguish between the complexed and noncomplexed forms in a mixture due to the differences in their relative diffusion coefficient values. The lowest diffusion was attributed to the complexed species because of the increase in both size and molecular weight when compared with the noncomplexed forms. Mixtures of sugars of the same molecular weight and also the isomers of a single monosaccharide can be 'separated' by DOSY on the basis of their different tendencies to form complexes with different diffusion coefficient values.     

Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms

Schistosoma mansoni is a blood-dwelling trematode which can persist for several years in the vessels of the human host. The schistosomal surface has been extensively characterized by lectin binding studies, revealing the carbohydrate composition of the worm's tegument. Using fluorescent and scanning electron microscopy we demonstrate that the surface carbohydrates of cercariae and adult worms are the binding ligands for mannanbinding lectin (MBL), a serum protein that is part of the innate immune system. An in vitro complement activation assay with C1q-deficient complement suggests that MBL, in association with the serine proteases MASP-1 and MASP-2, is capable of fixing complement components on the schistosomal tegument and activating the complement cascade via the "MBL pathway." MBL is constitutively expressed by hepatocytes and present in the blood at a stable level. Since it is also a weak acute-phase protein and therefore upregulated in an acute-phase response we investigated the serum MBL levels in patients infected with Schistosoma sp. and in healthy control persons. An enzyme-linked immunosorbent assay indicated no differences between the two groups. Although our results suggest an involvement of MBL activated complement in vitro, its role in vivo remains to be clarified.