Proteolysis in hyperthermophilic microorganisms

Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.     

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.     

Healthy aging: the Swiss experience

The quantitative importance of ageing both in developed or in developing countries raised the subtle question of the quality of life of the ageing person. Precise definitions of life expectancy, healthy life expectancy and quality of life are presented. Then, presentation of the results of two cross-sectional studies performed with the same methodology at a 15-year time interval in French-speaking Switzerland illustrates compression of morbidity theory, with increased longevity, increased good perception of health and increased quality of life. Moreover, quantitative data concerning the longevity impact of hospital geriatric care are presented from Geneva. The 4-year survival rate of over 85 year old women, discharged from the hospital for acute care, reached 48%. Explanations of these outstanding medical progresses on longevity and socio-economical challenges of extreme ageing are discussed in the world context.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

An anatomofunctional brain database

This study proposes a closer look at the neuropsychological method defined as the study of the neural bases of the behavioural and cognitive functions using an organisation-representation model for current data and knowledge of the brain, and the application of an anatomofunctional database. A Centre of Cognitive Anatomy (CAC) was set up for the collection and processing of neuronatomical, neuropsychological, and psycho-behavioural data for patients presenting sequels of focal brain damage. Such a system would allow concurrent treatment of anatomical and functional data. We would expect the results from such a model to produce stable 'anatomofunctional laws' that would be independent of all inter-individual variations in the functioning of the brain and could be checked against the entire database of information. A direct application would be the improvement of cognitive and/or behavioural rehabilitation of patients with brain damage.     

Effect of dietary fats on hepatic lipid metabolism in the growing turkey

The influence of dietary fatty acids on hepatic capacity of lipid synthesis and secretion was investigated in 7-week-old male turkeys. They were fed 10% of either lard (rich in saturated and monounsaturated fatty acids) or linseed oil (rich in polyunsaturated fatty acids, especially 18:3n-3). Fattening was identical with both diets (0.15-0.20% of abdominal adipose tissue), but the proportion of muscle Pectoralis major was lower with linseed oil (6.6 vs. 7.4%). Specific activities of lipogenic enzymes (ME, G6PDH, ACX, and Delta9-desaturase) were not influenced by the diet, however, FAS activity was lower with linseed oil (14.3 vs. 25.4 nM NADPH fixed/min). Fasting concentrations of lipoproteins synthesized and secreted by the liver, VLDL and HDL, were also lower with linseed oil, as well as plasma concentrations of phospholipids and cholesteryl esters. However, when VLDL catabolism was inhibited by injection of an antiserum against LPL, VLDL concentration was identical in both groups (100-120 mg/l), whereas that of phospholipids and cholesteryl esters, that are transported by HDL mainly, remained lower with linseed oil. Thus, in the growing turkeys, and contrary to mammals and the chicken, feeding n-3 polyunsaturated fatty acids did not decrease hepatic triglyceride synthesis and secretion, nor fattening. By contrast, in this species, n-3 polyunsaturated fatty acids appear to influence mostly HDL metabolism, with a negative impact on muscular growth.     

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.     

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells

The bioluminescent Ca²⁺-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca^{2 +}-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC₅₀ value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca^{2 +}-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I. Revised version received: 12 September 2001 Electronic Publication