Protection of Mice against Lethal Coxsackievirus B3 Infection by Using DNA Immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.     

Cryotolerance and Cold Adaptation in Lactococcus lactis Subsp. lactis IL1403

The physiology of the cold-shock response in Lactococcus lactis subsp. lactis IL1403 at a subzero temperature, and cold-induced adaptation to heat shock, were investigated. Preincubation of cells at 8°C led to the development of cryotolerance, i.e., an enhanced capacity to survive exposure to freezing temperature (-20°C). Pretreatment with chemicals considered to be chaotropic agents did not induce cryotolerance or, in contrast, led to a decrease in survival capacity at -20°C. Interestingly, preincubation at 8°C led also to thermololerance to a 52°C challenge, but preincubation of cells at 42°C for 30 min did not improve their capacity to survive freezing-thawing exposure. These results demonstrate that cold- and heat-shock responses are physiologically linked by a complex relation. Furthermore, food processing at low temperature before subzero or heat treatment may need to be reconsidered.     

Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 ^-/- CD4⁺ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.     

Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon—a quantitative immunohistochemical study

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas’ disease.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.     

5 alpha-androstane-3 beta,17 beta-diol hydroxylating enzymes in stroma and epithelium of human benign prostatic hyperplasia (BPH)

As enzymatic hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) may be a factor in controlling the 5 alpha-dihydrotestosterone (DHT) content in the prostate, we were interested in activity and distribution of these enzymes in epithelium and stroma of human benign prostatic hyperplasia (BPH). The enzyme activities were measured after mechanical separation of BPH tissue from 15 patients of various ages into stroma and epithelium, and optimization of the in vitro transformation of 3 beta-diol to hydroxylated products, which were analyzed by HPLC. The main results were: (1) 3 beta-diol was hydroxylated at C-7 alpha, C-7 beta, C-6 alpha, and C-6 beta. (2) The mean Michaelis constant Km (nM +/- SEM) for hydroxylation at C-7 alpha(beta) (168 +/- 21) was significantly lower than at C-6 alpha(beta) (601 +/- 43) without differences between stroma and epithelium. (3) Hydroxylation at alpha position dominated significantly over that at beta. (4) The mean maximal metabolic rate Vmax (pmol . mg protein-1 . h-1) of hydroxylation at C-6 alpha was about 7-fold lower in stroma (3.4 +/- 0.2) than in epithelium (23.8 +/- 4.1), concerning the other hydroxylations, Vmax was about 1.6-fold lower in stroma. (5) With increasing age of the patients there was a significant decrease of the 3 beta-diol hydroxylation in stroma and epithelium. It is discussed that the significantly lower activity of 3 beta-diol hydroxylation in stroma compared to epithelium and the decrease of activity with increasing age might potentiate the DHT accumulation in stroma of BPH.