Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products

Proteolytic activity and the cysteine protease profile were determined for enzymatic extracts (EE) from Carica papaya L. cv. Maradol harvest by-products (stems, unripe fruit, petioles and leaves). The proportion of each by-product type in the sampled plantation was calculated. Polypeptide bands were identified by SDS–PAGE for each EE and molecular weight calculated for the cysteine proteases. Leaf and fruit tissue had the highest protein contents of the by-products. Leaf tissue also produced the highest total EE yield. All the SDS–PAGE gels for the EE’s exhibited an approximately 23 kDa band probably corresponding to papain. The zymography profiles of the EE’s were similar, with bands at approximately >202.8, 76.8, 55.4 and 46.5 kDa. The fruit EE had the highest specific proteolytic activity and the leaf EE the lowest. Fruit and stem by-products are the most promising for proteolytic enzyme extraction.     

Analysis of NK cells and chemokine receptors in tumor infiltrating CD4 T lymphocytes in human renal carcinomas

Recent data suggest that chemokines and chemokine receptors mediate leukocyte recruitment of all components of the antitumor response. This study aimed to phenotypically characterize the immune lymphocyte infiltrate in human renal cell carcinomas (RCCs) and at the invasive margin (tumor–host interface) and to define the association of these findings with established prognostic indicators. Tumor infiltrating lymphocytes (TILs) were obtained from 24 patients with RCC undergoing radical nephrectomy. Peripheral blood cells from 37 patients were also obtained before surgery. Our findings are consistent with the preferential recruitment of CD4+ Th1-polarized effector memory cells that express CXCR3/CCR5. These cells were the main component of TILs and expressed as CXCR3, CCR5, CD45RO, and CD95. Natural killer (NK) cells were found in significantly higher proportions in TILs of RCCs than in peripheral blood lymphocytes (PBLs) or in other tumors studied (colorectal and breast cancers), where these cells were found in small proportions. No differences in nuclear grade or other studied parameters were observed between the TILs and the lymphocytes present at the invasive margin, which showed a similar composition. However, differences were found according to the tumor stage. First, significantly fewer NK cells were observed in PBLs from metastatic patients. Second, a significantly lower proportion of CCR5/CXCR3/CD4+ cells and a higher proportion of CCR4/CD4+ cells were observed in metastatic patients, suggesting that preferential Th1-polarization may gradually change during the progression of renal cancer cells. Finally, the frequency of CD25/CD4+ cells was higher in metastatic patients. Although the sample of patients with metastasis was small, the overall results suggest a change in composition of the TILs that may potentially confer a selective advantage for tumor growth and may account for the suppression of an effective cytotoxic response.     

Microbiological control in stem cell banks: approaches to standardisation

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.     

Production of yam microtubers using a temporary immersion system

Yam clones ‚Pacala Duclos’ and ‚Belep’ in temporary immersion system culture showed favourable results on shoot growth stage and in the development of microtubers in comparison with solid culture media. Cultures in temporary immersion systems in both clones obtained a higher microtuber number per plant, with greater fresh weight and diameter in comparison with solid culture media. Besides, 45 and 47% of microtubers greater than 3.0 gFW for ‚Belep’ and ‚Pacala Duclos’ clones respectively, were obtained. Those tubers may be planted without acclimatization and may be stored for a prolonged period of time.     

Mapping sugar beet pectin acetylation pattern

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.     

Non-competitive inhibitors of metabotropic glutamate receptor 5 (mGluR5)

Based on a pharmacophore alignment on known non-competitive mGluR5 inhibitors applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)'s as low as around 100 nM were achieved.     

Prostate cancer outcome: epidemiology and biostatistics

Substantial gaps exist in our ability to accurately predict prognosis, and these gaps limit our understanding of the complex mechanisms that contribute to the greatest cancer epidemic of our time, prostate cancer. This review addresses contemporary epidemiologic and biostatistical issues in prostate cancer. It covers the science of outcome prediction and biomarker evaluation, recognition of the need to combine biomarkers to improve the accuracy of our outcome estimates and an analysis of current outcome assessment methods, including the TNM staging system and multivariate regression models. The simplicity and intuitive ease of the current TNM staging system must be balanced against its serious limitations in predictive accuracy and its loss of clinical utility. Statistical regression methods are required as we move to the new era of personalized medicine. We must implement statistical approaches that integrate the new molecular biomarkers with existing prognostic biomarkers to accurately predict which patients require treatment and to determine the optimal therapy.     

Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis

The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and beta(1,2) cyclic glucan. M. loti lpsbeta2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lpsbeta1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lpsbeta2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lpsbeta1 codes for a glucosyltransferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti beta(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus tenuis. Cgs mutant induces normal root hair curling but is unable to induce the formation of infection threads. M. loti cgs mutant was more sensitive to deoxycholate and displayed motility impairment compared with the wild-type strain. This pleiotropic effect depends on calcium concentration and temperature.