Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells

Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5-10min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.     

No evidence of fetal DNA persistence in maternal plasma after pregnancy

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.     

Preclinical evaluation of anti-CD86 immunotoxin in rhesus monkeys: analysis of systemic toxicity,pharmacokinetics, and effect on primary T-cell responses

CD86 and CD80 costimulatory antigens are highly overexpressed on Hodgkin/Reed-Sternberg cells in patients with Hodgkin's disease (HD) and are candidate target antigens for immunotoxins in order to eliminate minimal residual disease. In this study we have evaluated the pharmacokinetics (and immunological) toxicity in rhesus monkeys of immunotoxins consisting of gelonin conjugated to anti-CD86 (alphaCD86-IT). Both alphaCD86-IT and alphaCD80-IT inhibited protein synthesis in B cell lines from rhesus monkeys and inhibited the mixed leukocyte reaction. Reactivity of the alphaCD86 antibody with rhesus monkey CD86 (RhCD86) was shown by cloning and transfecting RhCD86, which conferred reactivity of the alphaCD86 antibody used in this study. alphaCD86-IT was administered as single intravenous bolus injection in four rhesus monkeys, and achieved plasma concentration in 50-fold excess able to eliminate cultured Hodgkin/Reed-Sternberg cells up to 6 h. The animals were capable of generating primary immune responses to both gelonin and murine IgG within 9 days after infusion with alphaCD86-IT. No evidence could be found of significant (immunological) toxicity. The results of this study show that alphaCD86-IT can be applied safely in an effective dose.     

Nutraceutical production with food-grade microorganisms

Over the past few years a number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. These include components that have a proven beneficial effect on human health, such as low-calorie sugars and B vitamins. Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food-grade microoganisms means that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.     

Three-dimensional organization of the glomeruli in the antennal lobe of the parasitoid wasps <Emphasis Type="Italic">Cotesia glomerata</Emphasis> and <Emphasis Type="Italic">C. rubecula</Emphasis>

Two closely related parasitoid wasp species, Cotesia glomerata and C. rubecula, differ in their use of associative learning. To investigate the neural basis underlying these differences, it is necessary to describe the olfactory pathway of both wasp species. This paper focuses on the organization of the glomeruli in the antennal lobe. Glomeruli were stained by retrograde axon tracing of all axons in the antennal nerve and observed by confocal laser scanning microscopy. Stacks of optical sections were processed with AMIRA software, and 3D digital models of the glomeruli were produced. The combined use of 2D images and 3D surface models of the antennal lobes enabled the identification of a set of corresponding glomeruli in both wasp species. This offers unique opportunities for the study of subtle differences involved in synaptic plasticity that may occur at the glomerular level and factors regulating this plasticity.     

Triggering of transient LES relaxations in ferrets: role of sympathetic pathways and effects of baclofen

Activation of gastric vagal mechanoreceptors by distention is thought to be the trigger for transient lower esophageal sphincter relaxations (TLESR), which lead to gastroesophageal reflux. The contribution of higher-threshold gastric splanchnic mechanoreceptors is uninvestigated. GABA(B) receptor agonists, including baclofen, potently reduce triggering of TLESR by low-level gastric distention. We aimed to determine first whether this effect of baclofen is maintained at high-level distention and second the role of splanchnic pathways in triggering TLESR. Micromanometric/pH studies in conscious ferrets showed that intragastric glucose infusion (25 ml) increased triggering of TLESR and reflux. Both were significantly reduced by baclofen (7 micromol/kg ip) (P < 0.05). When 40 ml of air was added to the glucose infusion, more TLESR occurred than with glucose alone (P < 0.01). These were also reduced by baclofen (P < 0.001). TLESR after glucose/air infusion were assessed before and after splanchnectomy (2-4, 9-11, and 23-25 days), which revealed no change. Baclofen inhibits TLESR after both low- and high-level gastric distention. Splanchnic pathways do not contribute to increased triggering of TLESR by high-level gastric distention.     

Combined action of S-carvone and mild heat treatment on Listeria monocytogenes Scott A

The combined action of the plant-derived volatile, S-carvone, and mild heat treatment on the food-borne pathogen, Listeria monocytogenes, was evaluated. The viability of exponential phase cultures grown at 8 degrees C could be reduced by 1.3 log units after exposure to S-carvone (5 mmol l-1) for 30 min at 45 degrees C, while individual treatment with S-carvone or exposure to 45 degrees C for 30 min did not result in a loss in viability. Other plant-derived volatiles, namely carvacrol, cinnamaldehyde, thymol and decanal, were also found to reduce the viability of L. monocytogenes in combination with the same mild heat treatment at concentrations of 1.75 mmol l-1, 2.5 mmol l-1, 1.5 mmol l-1 and 2 mmol l-1, respectively. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or other individual hurdles.     

Adaptation of the food-borne pathogen Bacillus cereus to carvacrol

Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations. A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol. Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells. Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C. The addition of carvacrol to cell suspensions of adapted B. cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol. During adaptation, changes in the fatty acid composition were observed. The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased. The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells. It could be concluded that B. cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.     

The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258/propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator/urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFalpha or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.