In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells

While caspases play an established role as intracellular executors of apoptosis, little is known about extracellular activities of this ubiquitously expressed family of proteases. We demonstrate here that recombinant caspase-3 retained enzymatic activity in various extracellular fluids. Experiments with cell lines, primary cells, and mice with fulminant CD95-triggered hepatitis showed that significant amounts of DEVD-aminofluoromethylcoumarine-cleaving activity, indicative of active effector caspases, were released into the medium/plasma during apoptosis. Furthermore, caspase activities were detected in liquor samples from human head trauma patients. These findings warrant closer investigation of DEVDase activity as a diagnostic marker, and of potential extracellular substrates for caspases.     

Galactonate determination in urine by stable isotope dilution gas chromatography-mass spectrometry

A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.     

Relationships between the flexed-arm hang and select measures of muscular fitness

The flexed-arm hang (FAH) has been used to assess arm and shoulder girdle strength for 35 years despite little evidence to support its use. The purpose of this study was to determine what muscular fitness component, if any, was related to the FAH. The traditional overgrip chin-above-bar test and 5 different variations were compared with absolute strength (1 repetition maximum [1RM] lat pull down), relative strength (1RM.mass(-1)), and muscle endurance (repetitions to failure at 70% of the 1RM). Sixty college-age women volunteered for the study. Relationships were examined using Pearson Product Moment Correlation. No significant relationship was found between any of the FAH variations and absolute strength or muscle endurance; however, all FAH variations correlated significantly with relative strength (1RM.mass(-1)). The strongest relationship was with the undergrip FAH timed to 90 degrees of elbow extension (r = 0.72). Investigators concluded that the FAH is a test of weight-relative muscular strength and appears unrelated to absolute strength or muscle endurance.     

Cryptic repeated genomic recombination during speciation in Gossypium gossypioides

The Mexican cotton Gossypium gossypioides is a perplexing entity, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other American cottons. We reevaluated the evolutionary history of this enigmatic species using 16.4 kb of DNA sequence. Phylogenetic analyses show that chloroplast DNA (7.3 kb), nuclear ribosomal internal transcribed spacers (ITS; 0.69 kb), and unique nuclear genes (8.4 kb) yield conflicting resolutions for G. gossypioides. Eight low-copy nuclear genes provide a nearly unanimous resolution of G. gossypioides as the basalmost American diploid cotton, whereas cpDNA sequences resolve G. gossypioides deeply nested within the American diploid clade sister to Peruvian G. raimondii, and ITS places G. gossypioides in an African (rather than an American) clade. These data, in conjunction with previous evidence from the repetitive fraction of the genome, implicate a complex history for G. gossypioides possibly involving temporally separated introgression events from genetically divergent cottons that are presently restricted to different hemispheres. Based on repetitive nuclear DNA, it appears that G. gossypioides experienced nuclear introgression from an African species shortly after divergence from the remainder of the American assemblage. More recently, hybridization with a Mexican species may have resulted in cpDNA introgression, and possibly a second round of cryptic nuclear introgression. Gossypium gossypioides provides a striking example of the previously unsuspected chimeric nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events.     

Feast and famine in plant genomes

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a one-way ticket to genomic obesity, as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.     

Comparative studies of the adsorption behavior of plasma proteins to heparin-coated surfaces]

Over the past 10 years, investigations have shown that heparin coating optimizes haemocompatibility in extracorporeal circulation systems. To date, however, the mechanisms involved have not been identified. 20 ml of fresh human blood were circulated in an in vitro closed-loop model with and without heparin coating. Using a newly developed ELISA, a quantitative analysis of the adsorbed plasma proteins was done. In addition, changes in coagulation, complement and blood cell releasing factors were measured by ELISA methods: prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin-III complex (TAT), PMN-elastase, beta-thromboglobulin (beta-TG) and terminal complement-complex (TCC). In uncoated tubing, high concentrations of fibrinogen, fibronectin, prothrombin, vitronectin, alpha 2-macroglobulin, von Willebrand factor and complement factor C3 were found. Large amounts of antithrombin-III, HMWK and C1-esterase inhibitor were found on the heparinized surfaces. In addition, the concentrations of the soluble plasma protein markers F1 + 2, FPA, PMN-elastase, TCC and beta-TG were significantly lower (p < 0.05) in the presence of heparin coating. The haemocompatibility of "foreign" surfaces depends largely on the extent to which the processes of activation and inhibition of humoral and cellular mediators permanently occurring at the natural endothelium can be simulated. Owing to the low adsorption of procoagulatory and proinflammatory enzymes, heparin-coated surfaces demonstrate significantly improved haemocompatibility.     

Variation in the Bemisia tabaci s. l. species complex (Hemiptera: Aleyrodidae) and its natural enemies leading to successful biological control of Bemisia biotype B in the USA

Parasitoids of the Bemisia tabaci (Gennadius) species complex collected in Spain and Thailand were evaluated as biological control agents of B. tabaci biotype B in cole crops in Texas, USA. Parasitoids were identified by morphological and RAPD-PCR analyses. The most abundant parasitoid from Spain was Eretmocerus mundus Mercet with apparent field parasitism of 39-44%. In Thailand, Encarsia formosa Gahan, E. transvena Timberlake, E. adrianae Lopez-Avila, Eretmocerus sp. 1 and sp. 2 emerged, with apparent field parasitism of 1-65%. Identification and molecular classification of B. tabaci associated with parasitoid collections and in the release site in Texas were accomplished using morphological traits and nucleotide sequence comparison of the mitochondrial cytochrome oxidase I gene (COI) (700-720 bp). Collections of B. tabaci from Thailand grouped separately from B types from Arizona and Florida and the target B type from Texas, USA, a cluster from India, and other New World B. tabaci. The Spanish B. tabaci host of E. mundus which was laboratory and field-tested to achieve biological control of the B type was most closely related to non-B type B. tabaci populations from Spain and Sudan, the latter which formed a second group within the larger clade that also contained the B type cluster. Laboratory tests indicated that E. mundus from Spain parasitized more B. tabaci type B than did Eretmocerus spp. native to Texas and other exotic parasitoids evaluated. Eretmocerus mundus from Spain also successfully parasitized B. tabaci type B when field-released in a 0.94 million ha test area in Texas, and has significantly enhanced control of B. tabaci type B in California, USA. In contrast, parasitoids from Thailand failed to establish in the field in Texas, collectively suggesting a positive correlation between the centres of diversity of compatible parasitoid-host complexes.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Identification of Predictive Early Biomarkers for Sterile-SIRS after Cardiovascular Surgery

Systemic inflammatory response syndrome (SIRS) is a common complication after cardiovascular surgery that in severe cases can lead to multiple organ dysfunction syndrome and even death. We therefore set out to identify reliable early biomarkers for SIRS in a prospective small patient study for timely intervention. 21 Patients scheduled for planned cardiovascular surgery were recruited in the study, monitored for signs of SIRS and blood samples were taken to investigate biomarkers at pre-assigned time points: day of admission, start of surgery, end of surgery, days 1, 2, 3, 5 and 8 post surgery. Stored plasma and cryopreserved blood samples were analyzed for cytokine expression (IL1β, IL2, IL6, IL8, IL10, TNFα, IFNγ), other pro-inflammatory markers (sCD163, sTREM-1, ESM-1) and response to endotoxin. Acute phase proteins CRP, PCT and pro-inflammatory cytokines IL6 and IL8 were significantly increased (p<0.001) at the end of surgery in all patients but could not distinguish between groups. Normalization of samples revealed significant increases in IL1β changes (p<0.05) and decreased responses to endotoxin (p<0.01) in the SIRS group at the end of surgery. Soluble TREM-1 plasma concentrations were significantly increased in patients with SIRS (p<0.01). This small scale patient study could show that common sepsis markers PCT, CRP, IL6 and TNFα had low predictive value for early diagnosis of SIRS after cardiovascular surgery. A combination of normalized IL1β plasma levels, responses to endotoxin and soluble TREM-1 plasma concentrations at the end of surgery are predictive markers of SIRS development in this small scale study and could act as an indicator for starting early therapeutic interventions.     

The Open AUC Project

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software.