Prognostic impact of B-cell density in cutaneous melanoma

Studies on the prognostic importance of tumor-infiltrating lymphocytes have mainly focused on T cells, while little is known about the role of tumor-infiltrating B lymphocytes. We investigated the prevalence of CD20⁺ B cells by immunohistochemistry in primary melanoma samples of 106 patients and analyzed in relation to clinicopathological parameters and patients’ survival. The majority of samples contained a significant amount of B lymphocytes, predominantly dispersed in the stroma surrounding tumor deposits (mean peritumoral and intratumoral densities: 178.7 ± 156.1 vs. 4.9 ± 6.9 cells/mm², respectively). B cells organized in follicle-like aggregates were also observed in 26% of the samples. B-cell density correlated with that of activated (CD25⁺ or OX40⁺) T lymphocytes. Infiltration by CD20⁺ lymphocytes did not correlate with tumor thickness, while the presence of B-cell aggregates was observed more frequently in thick melanomas. On the other hand, B-cell infiltration was more pronounced in nonmetastatic or lymph node metastatic tumors, compared to visceral metastatic ones. Accordingly, high number of these cells provided significant survival advantage (P = 0.0391 and P = 0.0136 for intra- and peritumoral infiltration, respectively). Furthermore, combination of peritumoral B-cell density with the number of activated T lymphocytes identified patient subgroups with different disease outcome, which was most favorable in the case of high density, while very poor in the case of low density of both cell types. Multivariate survival analysis identified tumor thickness and CD20⁺/OX40⁺ cell density combination as significant independent prognostic factors. Taken together, our results show correlation between low number of CD20⁺ B lymphocytes and melanoma progression, indicating a possible role of tumor-infiltrating B cells in antitumoral immune response. It was also reflected in better outcome of the disease since the density of B lymphocytes alone as well as in combination with that of activated T cells proved of prognostic importance in patients with malignant melanoma.     

How does hydroxyl introduction influence the double helical structure: the stabilization of an altritol nucleic acid:ribonucleic acid duplex

Altritol nucleic acids (ANAs) are a promising new tool in the development of artificial small interfering ribonucleic acids (siRNAs) for therapeutical applications. To mimic the siRNA:messenger RNA (mRNA) interactions, the crystal structure of the ANA:RNA construct a(CCGUAAUGCC-P):r(GGCAUUACGG) was determined to 1.96?? resolution which revealed the hybrid to form an A-type helix. As this A-form is a major requirement in the RNAi process, this crystal structure confirms the potential of altritol-modified siRNAs. Moreover, in the ANA strands, a new type of intrastrand interactions was found between the O2' hydroxyl group of one residue and the sugar ring O4' atom of the next residue. These interactions were further investigated by quantum chemical methods. Besides hydration effects, these intrastrand hydrogen bonds may also contribute to the stability of ANA:RNA duplexes.     

Ligand chirality effects on the dynamics of human 3-phosphoglycerate kinase: comparison between D- and L-nucleotides

l-nucleoside analogues are now largely used as antiviral drugs for the treatment of viral infections like HBV, HCV and HIV. However, in order to be fully active, they need to be phosphorylated by cellular or viral kinases. Human 3-phosphogycerate kinase (hPGK) was shown to catalyze the last step of activation of l-enantiomers and thus constitutes an attractive target for theoretical predictions of its phosphorylation efficiency. Molecular dynamics simulations were carried out with four different nucleotides (d-/l-ADP and d-/l-CDP) in complex with hPGK and 1,3-bisphospho-d-glycerate (bPG). The binding affinities of CDPs (both enantiomers) for hPGK were found very weak while d- and l-ADP were better substrates. Interestingly, the binding affinity of the bPG substrate was found to be lower in presence of d-ADP than l-ADP which indicates a potential antagonistic effect on one substrate to the other. A detailed analysis of the simulations unravels important dynamic conditions for efficient phosphorylation. Indeed, as previously described for the natural substrate, the hinge bending motion of the domains upon substrates binding should be more correlated and directional. Interestingly, the unforeseen finding was the larger dynamics freedoms observed for the substrates that was favored by the protein atoms flexibility around the nucleobase binding site.     

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.     

Morphological events in cultures of mechanically isolated maize mcirospores

Summary In tis androgenic response, maize is considered to be a recalcitrant plant. We used mechanically isolated microspores of maize genotype A18 to establish a responsive microspore culture of maize. Morphological events occurring during the first days of maize androgenesis in a microspore culture were observed and described, and some morphological markers for distinguishing between embryogenic microspores and nonembryogenic microspores were identified. It was found that the enlargement of microspores during the first days in culture and the ‘star-like’ organization of the cytoplasm inside the microspore are connected with reprogramming of the developmental pathway in maize microspores. Some differences were also found in the surface wall architecture of embryogenic microspores. Fertile plants were successfully recovered from microspore-originated structures.     

Different Desensitization Patterns for Sensory and Vascular TRPV1 Populations in the Rat: Expression,Localization and Functional Consequences

Background and purpose

TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known.

Experimental approach

Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry.

Key results

Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle.

Conclusion and implications

Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.     

Detection of the Bcl I polymorphism of the glucocorticoid receptor gene by single-tube allele-specific polymerase chain reaction

The Bcl I polymorphism of the glucocorticoid receptor gene, recently identified as an intronic C to G change 646 nucleotides downstream of exon 2, has been associated with increased sensitivity to glucocorticoids and its potential relevance in metabolic disturbances and in various disorders has been extensively investigated. In the present study, we designed a single-tube allele-specific polymerase chain reaction for genotyping this polymorphism in peripheral blood DNA samples. When the Bcl I polymorphism was detected with this novel method in a cohort of 247 healthy subjects, the observed genotype distribution matched the Hardy–Weinberg equilibrium (100 subjects homozygous for the wild-type, 124 heterozygous and 23 homozygous for the mutant allele). In 50 randomly selected subjects the Bcl I polymorphism was also determined using a traditional restriction fragment length polymorphism technique and DNA sequencing, and the results showed 100% coincidence with those obtained by our novel method. The method proved to be more rapid and less labour-intensive compared to currently used techniques, and it avoided the use of extensive instrumentals. We assume that this novel method may have a broad utility in clinical and molecular epidemiological studies aimed to elucidate the impact of the Bcl I polymorphism of the glucocorticoid receptor gene either on metabolic disturbances, or various disorders, including cancer treatment and hormone substitution therapies.     

Susceptibility of dopamine D5 receptor targeted mice to cysteamine

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.