BCG immunotherapy in stage I melanoma patients

Summary Previously, we have provided evidence for a positive correlation between HLA-DR expression in primary melanoma and early metastasis [3, 4]. In the present study we investigated whether this relationship was modified by adjuvant BCG immunotherapy. The study comprised 107 patients with a stage I high-risk melanoma; 44 patients had been treated with BCG, whereas the remaining patients had not received any adjuvant therapy. There was no difference in disease-free survival between BCG-treated and untreated patients. Disease-free survival was significantly shorter in patients with high expression of HLA-DR antigens in the primary tumor.Subgrouping BCG-treated and control patients according to HLA-DR phenotype of the melanoma revealed a prolongation of disease-free survival in the subgroup of BCG-treated patients with no or low expression of HLA-DR antigens in the primary melanoma. BCG therapy apparently did not influence prognosis of patients with high expression of HLA-DR antigens in the tumor.     

Predicted membrane topology of the coronavirus protein E1

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.     

Cholesteryl ester transfer protein and hyperalphalipoproteinemia in Caucasians

It is unclear whether cholesteryl ester transfer protein (CETP) contributes to high density lipoprotein cholesterol (HDL-C) levels in hyperalphalipoproteinemia (HALP) in Caucasians. Moreover, even less is known about the effects of hereditary CETP deficiency in non-Japanese. We studied 95 unrelated Caucasian individuals with HALP. No correlations between CETP concentration or activity and HDL-C were identified. Screening for CETP gene defects led to the identification of heterozygosity for a novel splice site mutation in one individual. Twenty-five heterozygotes for this mutation showed reduced CETP concentration (-40%) and activity (-50%) and a 35% increase of HDL-C compared with family controls. The heterozygotes presented with an isolated high HDL-C, whereas the remaining subjects exhibited a typical high HDL-C/low-triglyceride phenotype. The increase of HDL-C in the CETP-deficient heterozygotes was primarily attributable to increased high density lipoprotein containing apolipoprotein A-I and A-II (LpAI:AII) levels, contrasting with an increase in both high density lipoprotein containing apolipoprotein A-I only and LpAI:AII in the other group. This study suggests the absence of a relationship between CETP and HDL-C levels in Caucasians with HALP. The data furthermore indicate that genetic CETP deficiency is rare among Caucasians and that this disorder presents with a phenotype that is different from that of subjects with HALP who have no mutation in the CETP gene.     

Mechanism of flavin reduction in the class 1A dihydroorotate dehydrogenase from Lactococcus lactis

Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate (OA) using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (cysteine for class 1A DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for the class 1A enzyme from Lactococcus lactis by determining kinetic isotope effects (KIEs) on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined at two pH values. At pH 7.0, KIEs were approximately 2-fold for DHO labeled singly at the 5-position or the 6-position and approximately 4-fold for DHO labeled at both the 5- and 6-positions. At pH 8.5, the KIEs observed for DHO labeled at the 5-position, the 6-position, and the 5- and 6-positions were approximately 2-, approximately 3-, and approximately 6-fold, respectively. These isotope effects are consistent with a concerted oxidation of DHO. The pH dependence of reduction was also determined, and a pKa of 8.3 was found. This pKa can be attributed to the ionization of the active site cysteine which deprotonates C5 of DHO during the reaction. To further investigate the importance of the active site base, two site-directed mutants were also studied: Cys130Ala (removal of the active site base) and Cys130Ser (replacement with the active site base used by class 2 DHODs). Both mutant enzymes exhibited binding affinities for DHO similar to that of the wild-type enzyme. Reduction of both mutants was extremely slow compared to that of the wild type; the rate of reduction increased with pH, showing no sign of a plateau. Interestingly, double-deuterium isotope effects on the Cys130Ser mutant also showed a concerted mechanism for flavin reduction.     

Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731), its aglycones and structurally related compounds

The special extract ERr 731^® from the roots of Rheum rhaponticum is the major constituent of Phytoestrol^® N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731^® was unknown. For the first time, ERr 731^® and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor- or estrogen receptor-β (ER, ERβ). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17β-estradiol or the selective ER-(propylpyrazoltriol) or ERβ-agonists (diarylpropionitril) were used. Neither in ER-expressing yeast cells, in the ER-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ER an activation of ER by ERr 731^® or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERβ, 100 ng/ml ERr 731^® and the single compounds significantly induced the ERβ-coupled luciferase activity in a range comparable to 10⁻⁸ M 17β-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERβ agonistic activity by ERr 731^® could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERβ activation.     

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC 33499). The DNA-DNA hybridization revealed a similarity of 66% with E. adhaerens (NCIMB12342(T)). In contrast to the type strain the isolate M3A possesses the capacity to nodulate plant roots. This is the first report on the detailed identification of a rhizobia-related strain from the intestinal tract of animals. Strain M3A has been deposited with two culture collections (DSM10169; ATCC BAA-396).     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Phylogenetic relationships of finches and allies based on nuclear and mitochondrial DNA

The complete mitochondrial gene cytochrome b in combination with a nuclear gene, beta-fibrinogen intron 7, is sequenced for different groups of mostly granivorous species in the superfamily Passeroidea, with a focus on the estrildids and fringillids. From our study we can conclude that within the group of granivorous finches two clades can be distinguished, the estrildid weaver clade and the cardueline, fringillid, emberizid, passerine sparrow clade. In contrast to many other studies the passerine sparrows are not placed within the weavers estrildid clade which is in agreement with other recent studies (e.g., ). Our study also shows that the estrildids do form a monophyletic group, but there is a division based on geographic origin: an African group and an Asian-Australian group. Within the Fringillidae the Fringilla species are the sister group of the carduelines.