Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Bacterial l-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig l-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by l-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for l-asparaginases.     

IL-15 regulates homeostasis and terminal maturation of NKT cells

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.     

Low adiponectin serum level--reduced protective effect on the left ventricular wall thickness

Adiponectin, secreted by fat tissue, is down - regulated in obesity and may be involved in obesity-related disorders. It has anti-inflammatory, antiatherosclerotic and antidiabetic effect. Obesity is a strong predictor for hypertension and cardiovascular diseases. Recent studies showed that adiponectin level has important role in metabolic disorders, arterial hypertension and ischemic heart disease but its effect on left ventricular hypertrophy (LVH) has not been fully clarified. The aim of this research is to determine whether the protective effect of adiponectin against development of left ventricular hypertrophy is decreased in hypertensive overweight patients. The study included 61 adult, overweight hypertensive patients, with body mass index in range 25-30 kg/m2. Patients had regular morning glucose serum values and regular creatinine level. They were divided into four groups, according to sex and the presence of LVH. There were 16 female and 15 male hypertensive patients with LVH and 15 female and 15 male hypertensive patients without LVH, who were a control group. Glucose profile, lipidogram, creatinine clearance and anthropometric measures were determined in all patients. Cardiovascular measurements were taken applying two-dimensional ultrasound. Adiponectin serum level was measured using enzyme immunoassay (ELISA). Results showed that adiponectin serum level was significantly lower in hypertensive, overweight females and males with LVH than in the control groups without LVH. Adiponectin serum level did not correlate significant with intraventricular or with posterior wall thickness of left ventricle. Hypoadiponectinemia presents part of neurohumoral, non-haemodynamic system who contributes to obesity-related hypertension and left ventricular hypertrophy development. Low adiponectin level together with others adipokines, cytokines and chemokines secreted by fat tissue could contribute to pathophysiologic changes of the myocardium via unknown molecular mechanisms yet.     

Association of behavioral cardiovascular risk factors with mortality in Croatian adult population: the CroHort study

This study examined individual and combined influence of smoking, physical inactivity, alcohol drinking, and unhealthy diet on total mortality. Relationship between individual and combined poor health behaviours and total mortality were examined using Cox proportional hazards regression. Out of 7490 individuals included in the study, during 5 years follow up 808 died. Adjusted hazard ratios (HRs), and 95% confidence intervals (95% CIs) for men with health behaviour scores 1, 2, 3, and 4 compared with those with score 0 were 1.67 (1.24-2.24), 2.28 (1.64-3.18), 2.24 (1.32-3.84), and 2.86 (0.77-11.70), respectively (p value for trend < 0.001). Adjusted HRs (95% CIs) for women with health behaviour scores 1, 2, and 3 compared with those with score 0 were 1.17 (0.97-1.42), 1.37 (1.02-1.86), and 1.20 (0.37-3.61), respectively (p value for trend = 0.04). A unit of the health behaviour score increased mortality risk equivalent to being 5.9 and 2.9 years older, for man and woman respectively.     

Abbreviated Exposure to Hypoxia Is Sufficient to Induce CNS Dysmyelination,Modulate Spinal Motor Neuron Composition,and Impair Motor Development in Neonatal Mice

Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2–3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.     

Risk factors for cardiovascular disease in rural area of Croatia

Rural areas, where 47.6% of the Croatian population lives are not generally the focus of research; yet there are challenges which affect the rural population that often go unreported. These communities often exhibit disadvantages in many areas of health. The aim of this study is to examine the specific health needs and related determinants of rural populations influenced by transition that were affected by the consequences of war. The focus of the research is rural lifestyle, behaviour and cardiovascular risk factors in three villages of Sisacko-moslavacka County. Results show that participants generally understand that their own lifestyles influence their health, but they often neglect to change their behaviour to improve their health. This can be explained through complex socio-economic conditions and traditional values of their heritage. These results suggest a need for further research on health status, attitude, and behaviour of Croatia's rural population. Specific public health intervention and services for rural populations must be promoted.     

Relevance of the ability of fructose 1,6-bis(phosphate) to sequester ferrous but not ferric ions

The cytoprotective activity of F16BP has been documented in severe conditions such as convulsions, reperfusion injury, septic shock, diabetic complications, hypothermia-induced injury, UV-provoked skin damage and in other processes including apoptosis and excitotoxicity. F16BP shows very efficient cytoprotective activity in astroglial cells exposed to H₂O₂-provoked oxidative stress and during neuronal injury caused by hypoxic conditions. As most of the aforementioned processes involve iron activity-related conditions, we investigated the ferric and ferrous iron binding properties of F16BP under physiological conditions using ³¹P NMR and EPR spectroscopy. Our results indicate that cytoprotective F16BP activity is predominantly based on ferrous iron sequestration. ³¹P NMR spectroscopy of F16BP employing paramagnetic properties of iron clearly showed that F16BP forms stabile complexes with Fe²⁺ which was verified by EPR of another divalent cation—Mn²⁺. On the other hand, F16BP does not sequester ferric iron nor does it increase its redox activity as shown by ³¹P NMR and EPR spin-trapping. Therefore, F16BP may be beneficial in neurodegenerative and other conditions that are characterised by ferric iron stores and deposits.     

The expression of two engrailed-related genes in an apterygote insect and a phylogenetic analysis of insect engrailed-related genes

 Homologues of the Drosophila segment polarity gene engrailed have been cloned from many insect species, as well as other arthropods and non-arthropods. We have cloned partial cDNAs of two engrailed homologues, which we call engrailed-related genes, from the phylogenetically basal insect, Thermobia domestica (Order Thysanura) and possibly as many as four engrailed-related genes from the phylogenetically intermediate insect, Oncopeltus fasciatus (Order Hemiptera). Previous to our findings, only single engrailed-related homologues had been found in phylogenetically intermediate insect species (Tribolium and Schistocerca) and in the crustacean Artemia, while two engrailed-related homologues have been found in more derived orders (Hymenoptera and the engrailed and invected genes of lepidopterans and dipterans). Consequently, we performed a phylogenetic analysis of insect engrailed-related genes to determine whether insects ancestrally had one or two engrailed-related genes. We have found evidence of concerted evolution among engrailed-related paralogues, however, that masks the true phylogenetic history of these genes; the phylogeny may only be decipherable, therefore, by examining the presence or absence of engrailed-specific and invected-specific motifs, which will require cloning the full length cDNAs from more species. In addition, we examined the embryonic expression pattern of the two Thermobia engrailed-related genes; like Drosophila engrailed and invected, they are expressed in very similar patterns, but show one temporal difference in pregnathal segments that correlates with the tentative phylogenetic placement of the genes. Thermobia engrailed-related expression also confirms that the dorsal ridge is an ancient structure in insects. Received: 4 May 1998 / Accepted: 2 August 1998     

CytoITMprobe: a network information flow plugin for Cytoscape

ABSTRACT: BACKGROUND: Cytoscape is a well-developed flexible platform for visualization, integration and analysis of network data. Apart from the sophisticated graph layout and visualization routines, it hosts numerous user-developed plugins that significantly extend its core functionality. Earlier, we developed a network information flow framework and implemented it as a web application, called ITM Probe. Given a context consisting of one or more user-selected nodes, ITM Probe retrieves other network nodes most related to that context. It requires neither user restriction to subnetwork of interest nor additional and possibly noisy information. However, plugins for Cytoscape with these features do not yet exist. To provide the Cytoscape users the possibility of integrating ITM Probe into their workflows, we developed CytoITMprobe, a new Cytoscape plugin. FINDINGS: CytoITMprobe maintains all the desirable features of ITM Probe and adds additional flexibility not achievable through its web service version. It provides access to ITM Probe either through a web server or locally. The input, consisting of a Cytoscape network, together with the desired origins and/or destinations of information and a dissipation coefficient, is specified through a query form. The results are shown as a subnetwork of significant nodes and several summary tables. Users can control the composition and appearance of the subnetwork and interchange their ITM Probe results with other software tools through tab-delimited files. CONCLUSIONS: The main strength of CytoITMprobe is its flexibility. It allows the user to specify as input any Cytoscape network, rather than being restricted to the pre-compiled protein-protein interaction networks available through the ITM Probe web service. Users may supply their own edge weights and directionalities. Consequently, as opposed to ITM Probe web service, CytoITMprobe can be applied to many other domains of network-based research beyond protein-networks. It also enables seamless integration of ITM Probe results with other Cytoscape plugins having complementary functionality for data analysis.