A novel nucleotide found in human erythrocytes, 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, (1)H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 +/- 0.006 microm) in contrast with the high daily urine excretion of this compound (26.7 +/- 18.2 micromol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 +/- 0.321 microm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.     

Multijoint Control Strategies Transfer Between Tasks

In this paper, the hypothesis that multijoint control strategies are transferred between similar tasks was tested. To test this hypothesis, we studied the take-off phase of two types of backward somersault dives: one while translating backwards (Back), the other while translating forward (Reverse). An experimentally based dynamic model of the musculoskeletal system was employed to simulate the measured kinematics and reaction force data and to study the sensitivity of take-off performance to initial kinematic conditions. It was found that the horizontal velocity of the total body center of mass (CM) was most sensitive to modifications in the initial shank conditions. Consequently, the initial shank kinematics of the Back dive was modified in the optimization procedure while maintaining the joint coordination of the Back in order to generate the CM trajectory and reaction forces of a Reverse. Similarly, the initial shank kinematics of the Reverse dive was modified to simulate the CM trajectory and reaction force of the Back. It was found that small modifications in the initial shank kinematics led to change in direction of horizontal CM velocity at take-off; resulting in a switch from Back to Reverse and vice versa. In both cases, the simulated momentum conditions at departure and the bimodal shape of the reaction force-time curve were consistent with those experimentally observed. The results of this study support the hypothesis that transfer of control strategies between similar tasks is a viable option in multijoint control. This transfer of control strategy is explained using a hierarchical model of the motion control system.     

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.     

Angiogenic and anti-angiogenic factors in adrenal tumours

It appears that neoangiogenesis is an important factor in tumour invasion and the formation of metastases in several human cancers, and studies on pro-angiogenic and antiangiogenic factors are therefore of considerable interest to researchers. In this review we present pro-angiogenic and anti-angiogenic factors and other growth factors and their role in the formation of new blood vessels in adrenal tumours. Assessment of the angiogenic status of adrenal tumours and their vascular pattern may be useful for discriminating benign from malignant lesions and knowledge of their angiogenesis may be essential to the drawing up of promising treatment strategies for patients with malignant tumours.     

Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus

A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program—one susceptible (S), one resistant (R—that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.