Fluorescence resonance energy transfer detected by scanning near-field optical microscopy

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the F?rster mechanism over a range of 1-10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near-field optical microscope (SNOM) operated in the shared-aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel-by-pixel energy transfer efficiency measurements using SNOM.     

Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (DeltaE1) or lacking the Ad E3 region in addition to E1 sequences (DeltaE1DeltaE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34(cdc2) protein levels. In some instances, infection with DeltaE1 or DeltaE1 DeltaE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DeltaE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in DeltaE1 or DeltaE1 DeltaE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.     

Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

Five steps to selling cosmetic dentistry

Fischer 344 (F344) and Lewis (LEW) rats show considerable neuroanatomical and neurophysiological differences within the mesolimbic dopamine system. The aim of our experiments was to study the functional correlates of such differences by examining open-field behavior and the sensitivity towards the psychostimulant and rewarding effects of amphetamine in male and female, F344 and LEW rats. In addition, the consequences of short versus extended habituation to open-field testing on amphetamine locomotion in these two rat strains was assessed. LEW but not F344 rats irrespective of gender showed between-session habituation of open-field activity. Amphetamine-induced locomotion was higher in F344 compared to LEW rats and in females compared to male rats. In addition, extended habituation increased the locomotor effects of amphetamine. The rewarding effects of amphetamine as measured by the conditioned place preference test were more pronounced in F344 than in LEW rats. Our results suggest that the two rat strains differed in their behavioral response to mild stress and to amphetamine and that these differences may depend upon differences within the mesolimbic dopamine system.     

Gender comparison of peak oxygen uptake: repetitive box lifting versus treadmill running

The gender differences in peak oxygen uptake (VO2peak) for various modes of exercise have been examined previously; however, no direct gender comparisons have been made during repetitive lifting (RL). In the present study the VO2peak between RL and treadmill running (TR) was compared between 20 men [mean (SD) age, height, body mass and body fat: 21 (3) years, 1.79 (0.06) m, 81 (9) kg, 19 (6)%, respectively] and 20 women [mean (SD) age, height, body mass and body fat: 21 (3) years, 1.63 (0.05) m, 60 (7) kg, 27 (6)%, respectively]. VO2peak (l x min[-1]), defined as the highest value obtained during exercise to volitional fatigue, was determined using discontinuous protocols with treadmill grade or box mass incremented to increase exercise intensity. For RL VO2peak, a pneumatically driven shelf was used to lower a loaded box to the floor, and subjects then lifted the box, at a rate of 15 lifts x min(-1). VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and minute ventilation (VE, l x min[-1]) were determined using an on-line gas analysis system. A two-way repeated measures analysis of variance revealed significant gender effects, with men having higher values for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE, but women having higher values of the ventilatory equivalent for oxygen (VE/VO2). There were also mode of exercise effects, with TR values being higher for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE and an interaction effect for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE/VO2. The women obtained a greater percentage (approximately 84%) of their TR VO2peak during RL than did the men (approximately 79%). There was a marginal tendency for women to decrease and men to increase their VE/VO2 when comparing TR with RL. The magnitude of the gender differences between the two exercise modalities appeared to be similar for heart rate, VE and R, but differed for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]). Lifting to an absolute height (1.32 m for the RL protocol) may present a different physical challenge to men and women with respect to the degree of involvement of the muscle groups used during lifting and ventilation.     

Memory for duration: role of hippocampus and medial prefrontal cortex

In this task rats had to learn that a three-dimensional object stimulus (a rectangle) that was visible for 2 s would result in a positive (go) reinforcement for one object (a ball) and no reinforcement (no go) for a different object (a bottle). However, if the rectangle stimulus was visible for 8 s then there would be no reinforcement for the ball (no go), but a reinforcement for the bottle (go). After rats learned this conditional discrimination by responding differentially in terms of latency to approach the object, they received large (dorsal and ventral) lesions of the hippocampus, lesions of the medial prefrontal cortex (anterior cingulate and precentral cortex), lesions of the cortex dorsal to the dorsal hippocampus, or served as sham-operated controls. Following recovery from surgery they were retested. The results indicate that there were major impairments following hippocampal lesions, in contrast to cortical control and medial prefrontal cortex lesions, as indicated by smaller latency differences between positive and negative trials on postsurgery tests. In order to ensure that the deficits observed with hippocampal lesions were not due to a discrimination problem, new rats were trained in an object (gray cylinder) duration discrimination task. In this go/no go procedure, the rats were reinforced for a 2-s exposure (duration) of the gray cylinder, but not a 10-s duration, or vice versa. The results indicate that after hippocampal lesions, there was an initial deficit followed by complete recovery. There were no significant changes for the medial prefrontal, cortical control, or sham-operated animals. It appears that the hippocampus, but not the medial prefrontal cortex, is actively involved in representing in short-term memory temporal attribute information based on the use of markers for the beginning and end of the presence (duration) of a stimulus (object).     

Factors predicting a successful outcome after pharmacologic bowel compensation

OBJECTIVES: The authors determined those factors that predict a successful outcome in patients who receive pharmacologic agents to promote bowel absorption after massive intestinal resection. SUMMARY BACKGROUND DATA: Patients with the short bowel syndrome are maintained on long-term total parenteral nutrition (TPN) or more frequently considered for intestinal transplantation as part of their treatment program. The authors have administered a combination of trophic agents and a specialized diet to further enhance intestinal compensation and optimize nutrient absorption in patients with intestinal failure. METHODS: Forty-five TPN-dependent adults with a jejunal-ileal remnant < or = 50 cm and a portion of colon in continuity were treated with growth hormone, glutamine, and a modified diet for 4 weeks and observed for an average of 1.8 years. RESULTS: The average age of the patients was 43 years, the average jejunal-ileal length was 23 cm, and the average length of time the patient received TPN was 4.3 years. After 4 weeks of therapy, 26 (58%) were free of TPN support. Predictors of a favorable response included greater bowel length, lower body weight, and greater bowel length-body weight ratio. At follow-up, the percentage of patients who were not receiving TPN had fallen to 40%. CONCLUSIONS: Approximately half of a group of patients, thought to have absorptive surface area inadequate to be independent of TPN support, can maintain themselves on enteral feedings after this intestinal rehabilitation program. Because of the risk, costs, and alterations in lifestyle associated with long-term TPN or intestinal transplantation or both, it seems prudent to consider a program of bowel rehabilitation with an individual patient before embarking on another therapeutic plan.     

Vacuum punch biopsy under digital stereotaxic control, a new procedure in percutaneous diagnostic incision and excision biopsy of mammographic findings: initial experience

Schistosoma intercalatum bilharziasis continues to raise numerous questions regarding pathogenicity and gravity. The parasite was identified recently and the last fully described outbreak occurred 10 years ago in the city of Bata, Equatorial Guinea. Geographically Schistosoma intercalatum biharziasis is limited to one part of the African continent but has shown a tendency to spread. Hybridization of Schistosoma intercalatum and Schistosoma haematobium has been observed. The main clinical manifestation of Schistosoma intercalatum is rectal bleeding. The endoscopic appearance of lesions is variable and non-specific ranging from granulomas or polyps to ulcerations. Complications include severe rectitis or genital involvement such as salpingitis with secondary sterility. Spontaneous abortion has also been reported. Association with salmonella and klebsiella infection has been confirmed and can lead to life-threatening situations. Few studies have been performed to assess the value of diagnostic tests. The sensitivity of stool smears and urinary sedimentation testing is 81.7% and 56.3% respectively using the two examinations as references for one another. The sensitivity of immunological tests is generally good but varies depending on the reference technique used. Specificity can be affected by cross-reaction with other schistosomas or trematodes and even with nematodes and hematozoons. Treatment with a single dose of Biltricide has proven to be effective. Prevention requires education of the population at risk and use of molluscacides. The control strategy must be adapted in function of the epidemiology of the disease, diagnostic data, cost and effectiveness of screening and treatment.     

The prognostic significance of p34cdc2 and cyclin D1 protein expression in prostate adenocarcinoma

BACKGROUND: Cyclin-dependent kinases (CDK) and cyclins constitute the subunits of the maturation-promoting factor that controls the process of cell division. High levels of these proteins have been reported in human malignancies of the stomach, colon, breast, and lung, and have been implicated in aberrant cell division and dysregulated tumor growth. METHODS: p34cdc2 CDK and cyclin D1 (D1) protein expression were evaluated in 140 radical prostatectomy specimens harboring adenocarcinoma (PAC), using the respective monoclonal antibodies on archival tissue sections. In each case, slides stained with hematoxylin and eosin were examined for evaluation of Gleason's grade and pathologic stage. The DNA content of the tumors was determined by the Feulgen method with the CAS200 Image Analyzer (Cell Analysis Systems, Lombard, IL). Nuclear immunoreactivity for the two proteins was semiquantitatively scored, and results were correlated with Gleason's grade, stage, ploidy, metastatic status, and disease recurrence after radical prostatectomy. RESULTS: p34cdc2 was expressed in 84 of 140 PACs (60%) and correlated with high Gleason's grade (P = 0.0001), advanced pathologic stage (P = 0.01), nondiploid DNA content (P = 0.0001), and metastases (P = 0.04). On multivariate analysis using the Cox proportional hazards model, p34cdc2 immunoreactivity (P = 0.0001) and high Gleason's grade (P = 0.01) each independently predicted disease recurrence. When tumors were of low Gleason's grade and lacked p34cdc2 expression, 4 of 39 PACs (10%) recurred, as compared with 18 of 47 (38%) that recurred when tumors were of high Gleason's grade and expressed p34cdc2 protein. D1 was positive in 31 of 140 PACs (22%) and showed a trend (P = 0.07) of high Gleason's grade, but it did not reach statistical significance with any of the prognostic variables. In the majority of PACs expressing both p34cdc2 and D1 proteins, the adjacent benign prostate acini showed focal, scattered nuclear positivity of the basal and secretory epithelial cells. CONCLUSIONS: p34cdc2 is expressed in a majority of PACs and correlates with high Gleason's grade, advanced pathologic stage, nondiploid DNA content, and metastases. On multivariate analyses high Gleason's grade and p34cdc2 immunoreactivity predict disease recurrence independently of the pathologic stage. Thus, p34cdc2 appears to play a critical role in the evolution, proliferation, and spread of PACs and may be of prognostic value when applied to initial prostate tissue samples taken by needle biopsy.     

Serum interleukin 4 and interleukin 10 levels in patients with chronic hepatitis C virus infection

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.