Cellular entry and nuclear targeting by a highly anionic molecular umbrella

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.     

Improvements of TArgeted multiplex mass spectrometry IMaging

MALDI mass spectrometers have become popular tools for imaging histological sections. Currently this technology is primarily used for imaging naturally occurring molecules. Here we report on the improvement of TArgeted multiplex MS IMaging (TAMSIM) technology. For TAMSIM we attach photocleavable mass tags to antibodies. Staining histological sections is done analogously to standard immunohistochemical procedures with chemiluminescent or fluorescent detection with the sole difference that multiple antibodies each with a distinct mass tag are used in a single reaction. Mass tags are released from their respective antibodies by a laser pulse at 355 nm without added matrix. After scanning, MS images are created for each tag mass. The enhancements of TAMSIM presented here relate to four elements, the use of an improved generation of tags, their conjugation directly to primary antibodies, the comparison of fresh frozen sections with paraffin embedded ones for the TAMSIM imaging technology and finally, the increase of multiplex detection. Sections of healthy human pancreatic tissue were imaged to visualize different specific biomarkers (synaptophysin, chromogranin, insulin, calcitonin, somatostatin) in neuroendocrine cells of Langerhans islets. The aim was to localize these biomarkers on the tissue sections simultaneously.     

Seasonal phytoplankton responses to N:Si:P enrichment ratio in the Bizerte Lagoon (southwestern Mediterranean)

This study investigated the role of N, P and Si enrichments on phytoplankton in the Bizerte Lagoon (southwestern Mediterranean Sea, Tunisia) during March, June, August, October and December 2004. Polycarbonate bottles were enriched with different nutrients according to four treatments N:Si:P ratios [+NSi/-P (40:40:1), +P/-NSi (20:20:2,5), +NP/-Si (16:0:1) and +Si/-NP (16:32:1)] and incubated in situ during six days. Chl a and carbon biomass of phytoplankton varied significantly during the course of months, with the highest levels recorded in summer (4-4.4 microg Chl a L(-1) or 1126-1721 microg C L(-1)). Dinoflagellates dominated the initial phytoplankton communities, except in August, when diatoms represented a high fraction of microalgae (48%). Enrichment experiments induced significant increases in Chl a and in the final phytoplankton carbon biomasses. In summer (June/August), Si was the main limiting element for phytoplankton. Diatoms strongly responded to +Si/-NP and +NSi/-P enrichments and dominated the final phytoplankton communities (52-61%) in both treatments. Si played the most important role in the growth and development of diatoms. The biomasses and growth rates of dinoflagellates were significantly stimulated by +P/-NSi and +NP/-Si enrichments. After 6 days, dinoflagellates represented more than 70% of the total phytoplankton biomass in samples subjected to these treatments. Moreover, the addition of +P/-NSi increased the biomasses of several dinoflagellates. This suggests that dinoflagellates were mostly controlled by P availability. Unlike diatoms and dinoflagellates, flagellates showed weak responses to nutrient treatments during only some months of the year. The results showed that phytoplankton dynamics in the lagoon were influenced by nutrients in different manners.     

Monitoring of the adenovirus production process by flow cytometry

Adenovirus (Ad) has become the vector of choice for gene therapy clinical protocols worldwide; it is the only viral vector to date that has been licensed for use in a gene therapy treatment. There is, however, a need to develop a simple, reliable at-line method to monitor the production of virus and recombinant proteins (r-proteins) that have no intrinsic reporter properties. Here we utilize flow cytometry to measure cell size, granularity, and DNA content in a single-step analysis and to correlate these parameters to the production of a type-5 Ad (Ad5) expressing the recombinant green fluorescent protein (GFP). Clear correlations between these parameters and productivity are made, with forward scatter and DNA content showing the highest correlation coefficients, 0.9 and 0.83 for virus production and r-protein production, respectively. Measuring these parameters requires little or no processing of the cells from culture to analysis. These parameters have been used successfully to monitor, at-line, the amount of Ad and r-protein product in a 293-Ad system.     

Activation of cytosolic phospholipase A2alpha in resident peritoneal macrophages by Listeria monocytogenes involves listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.     

Chemopreventive effect of lycopene alone or with melatonin against the genesis of oxidative stress and mammary tumors induced by 7,12 dimethyl(a)benzanthracene in sprague dawely female rats

Breast cancer is the principle cause of death among women worldwide. In this study, we investigated the anti-tumor potential of lycopene (Lyco) alone or combined with melatonin (Lyco + Mel) for 120 days against a single oral dose of (50 mg/kg B.W.) 7,12-dimethylbenz(a)anthracene (DMBA)-induced oxidative stress and mammary carcinogenesis in female rats. The treatment protocol started from the day immediately after DMBA administration. Results obtained indicated that there was an elevation in the levels of malondialdhyde and nitric oxide in serum and breast tissues of DMBA injected rats. The combined treatment (Lyco + Mel) group showed a potential reduction of these parameters more than lyco individually. The activities of SOD, CAT, and GPx were found to be significantly high than lyco alone treated rats. In DMBA group a negative significant correlation between weight and serum nitric oxide (r = -0.59), and a positive significant correlation between NO and MDA (r = 0.81) was observed. Histopathological examination revealed the formation of tumor and angiogenesis in DMBA-induced rats and these abnormal changes were ameliorated by combined treatment with Lyco + Mel. In conclusion, these results suggested that supplementation of diet with lycopene with melatonin provided antioxidant defense with strong chemo preventive activity against DMBA-induced mammary tumors.     

Bacterial wilt and spot of tomato caused by <Emphasis Type="Italic">Xanthomonas vesicatoria </Emphasis>and <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in Egypt

Tomato fruits and seed lots were screened for the presence of Xanthomonas vesicatoria and Ralstonia solanacearum. Yellow colonies of Xanthomonas vesicatoria and white colonies of Ralstonia solanacearum were consistently isolated on yeast extract-dextrose-calcium carbonate agar medium (YDC) from diseased fruits and seed samples. This was confirmed by isolation on semi-selective medium such as Tween B for Xanthomonas and triphenyltetrazolium salt (TTC) medium for Ralstonia solanacearum followed by biochemical tests. The four isolates belonging to Xanthomonas vesicatoria and Ralstonia solanacearum were used to inoculate a local tomato variety. The isolates were found to cause yellowing and wilting of 2-weeks-old seedlings by 8–14 days after inoculation and by 4 weeks all plants had wilted and completely died. Bacteria with the same characteristics as those inoculated were reisolated from the infected plants. Uninoculated plants remained healthy.     

Ischemia-induced activation of AMPK does not increase glucose uptake in glycogen-replete isolated working rat hearts

Alterations in myocardial glucose metabolism are a key determinant of ischemia-induced depression of left ventricular mechanical function. Since myocardial glycogen is an important source of endogenous glucose, we compared the effects of ischemia on glucose uptake and utilization in isolated working rat hearts in which glycogen content was either replete (G replete, 114 micromol/g dry wt) or partially depleted (G depleted, 71 mumol/g dry wt). The effects of low-flow ischemia (LFI, 0.5 ml/min) on glucose uptake, glycogen turnover (glycogenolysis and glycogen synthesis), glycolysis, adenosine 5'-monophosphate-activated protein kinase (AMPK) activity, and GLUT4 translocation were measured. Relative to preischemic values, LFI caused a time-dependent reduction in glycogen content in both G-replete and G-depleted groups due to an acceleration of glycogenolysis (by 12-fold and 6-fold, respectively). In G-replete hearts, LFI (15 min) decreased glucose uptake (by 59%) and did not affect GLUT4 translocation. In G-depleted hearts, LFI also decreased initially glucose uptake (by 90%) and glycogen synthesis, but after 15 min, when glycogenolysis slowed due to exhaustion of glycogen content, glucose uptake increased (by 31%) in association with an increase in GLUT4 translocation. After 60 min of LFI, glucose uptake, glycogenolysis, and glycolysis recovered to near-preischemic values in both groups. LFI increased AMPK activity in a time-dependent manner in both groups (by 6-fold and 4-fold, respectively). Thus, when glycogen stores are replete before ischemia, ischemia-induced AMPK activation is not sufficient to increase glucose uptake. Under these conditions, an acceleration of glycogen degradation provides sufficient endogenous substrate for glycolysis during ischemia.