Suppressive Effect of Triethyllead on Entry of Proteins into the CNS Myelin Sheath In Vitro

Incorporation of [¹⁴C]leucine into the myelin sheath was studied in brain stem slices prepared from 22-day-old rats. Individual major myelin proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. There was a time lag before incorporation of the label into proteolipid protein (PLP) and intermediate protein (IP) reached maximal rates. Labelling of basic proteins (BP) and Wolfgram proteins (WP) revealed a much shorter lag in entry. Appearance of radioactive proteins in the myelin sheath was significantly hampered by triethyllead (PbEt₃) added to the incubation medium at micromolar concentrations. Inhibition values were highest in the case of PLP and were closely followed by the values for IP. BP and WP were less inhibited, although incorporation of these proteins into myelin was still suppressed more than was synthesis of total homogenate protein. Thus, myelin-forming cells seem to be unduly vulnerable to the toxin relative to the rest of the tissue. Furthermore, the results indicate an interference of PbEt₃ with certain posttranslational processes involved in furnishing of integral myelin proteins.     

Thymidine Kinase Diversity in Bacteria

Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.     

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.     

TNF-alpha-induced self expression in human lung endothelial cells is inhibited by native and oxidized alpha1-antitrypsin

Endothelial cells are among the main physiological targets of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In endothelial cells TNF-alpha elicits a broad spectrum of biological effects including differentiation, proliferation and apoptosis. alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases plays a vital role in protecting host tissue from proteolytic injury at sites of inflammation. Recently, it has been shown that AAT can be internalized by pulmonary endothelial cells, raising speculation that it may modulate endothelial cell function in addition to suppressing protease activity. Using Affymetrix microarray technology, real time PCR and ELISA methods we have investigated the effects of AAT on un-stimulated and TNF-alpha stimulated human primary lung microvascular endothelial cell gene expression and protein secretion. We find that AAT and TNF-alpha generally induced expression of distinct gene families with AAT exhibiting little activity in terms of inflammatory gene expression. Approximately 25% of genes up regulated by TNF-alpha were inhibited by co-administration of AAT including TNF-alpha-induced self expression. Surprisingly, the effects of AAT on TNF-alpha-induced self expression was inhibited equally well by oxidized AAT, a modified form of AAT, which lacks serine protease inhibitor activity. Overall, the pattern of gene expression regulated by native and oxidized AAT was similar with neither inducing pro-inflammatory gene expression. These findings suggest that inhibitory effects of native and oxidized forms of AAT on TNF-alpha stimulated gene expression may play an important role in limiting the uncontrolled endothelial cell activation and vascular injury in inflammatory disease.     

Do the unique properties of nanometals affect leachability or efficacy against fungi and termites?

Nanotechnology has the potential to affect the field of wood preservation through the creation of new and unique metal biocides with improved properties. This study evaluated leachability and efficacy of southern yellow pine wood treated with copper, zinc, or boron nanoparticles against mould fungi, decay fungi, and Eastern subterranean termites. Results showed that nanocopper with and without surfactant, nanozinc, and nanozinc plus silver with surfactant resisted leaching compared with metal oxide controls. Nearly all nanoboron and boric acid was released from the treated wood specimens during leaching. Mould fungi were moderately inhibited by nanozinc oxide with surfactant, but the other nanometal preparations did not significantly inhibit mould fungi. Mass loss from Gloeophyllum trabeum was significantly inhibited by all copper preparations, while Antrodia sp. was not inhibited by nanometal treatments. Nanometals imparted high resistance in southern yellow pine to the white-rot fungus, Trametes versicolor. Unleached specimens treated with nanoboron or nanozinc plus surfactant caused 100% and 31% mortality, respectively. All specimens treated with nanozinc or nanozinc plus silver inhibited termite feeding, but the copper treatments were less effective against termites. Nanozinc possessed the most favorable properties: leach resistance, termite mortality, and inhibition of termite feeding and decay by the white-rot fungus.     

Immigrational Background Affects the Effectiveness of a School‐based Overweight Prevention Program Promoting Water Consumption

We tested whether a simple overweight prevention program promoting water consumption in elementary schools is equally effective in children with an immigrational background (MIG) and in those without (non‐MIG). Thus, a secondary analysis of a controlled cluster trial, lasting one school year, was conducted. Thirty‐two elementary schools located in low socioeconomic districts in two German cities were included. Of the 2,950 school children analyzed, 1,306 were MIG children. Water fountains were installed in the schools of the intervention group (IG) and teachers held lessons to promote water consumption. Control schools (control group (CG)) did not receive any intervention. Before and after intervention, body weight and height was measured. Overweight was defined by age‐ and sex‐specific BMI cutoffs that are linked to an adult BMI of 25 kg/m². Beverage consumption was assessed in questionnaires. Modification of intervention effects by immigrational background was tested by interaction terms. The immigrational background modified the intervention effect on prevalence and remission of overweight (interaction term: P = 0.03 and P = 0.02), but not on the incidence of overweight (P = 0.06). After intervention, the risk of being overweight was reduced in the IG compared to the CG among non‐MIG (odds ratio = 0.51, 95% confidence interval (CI): 0.31–0.83), but not among MIG children (odds ratio = 1.02, 95% CI: 0.63–1.65). After intervention, water consumption significantly increased in the IG equally among both, non‐MIG and MIG, by ～1 glass/day. A simple school‐based intervention promoting water consumption prevented overweight in non‐MIG children, but failed in MIG children. Different beverage consumption, among other lifestyle factors, may account for this effect but scientific discussion remains open.     

A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.