Enhancement of the generation of reactive oxygen species may be involved in apoptotic pathway induction by 4HPR.

N-(4-Hydroxyphenyl)retinamide (4HPR) is currently used in cancer prevention and therapy trials. It is thought that its e ects result from induction of apoptosis. 4HPR-induced apoptosis in human cervical carcinoma C33A cells involves enhanced generation of reactive oxygen species (ROS). In this study we explored the mechanism by which 4HPR increases ROS and induces apoptosis in these cells. 4HPR induced cytochrome c release from mitochondria to cytoplasm, activated caspase-3, and caused a membrane permeability transition (MPT). All these 4HPR's e ects, as well as the induction of apoptosis, were inhibited by antioxidants, which decrease ROS. Thenoyltri¯uoroacetone, a mitochondrial respiratory chain (MRC) complex II inhibitor, and carbonylcyanide m-chlorophenyl hydrazone, which uncouples electron transfer and ATP synthesis and inhibits ROS generation by MRC, inhibited 4HPR-induced ROS generation very e ectively. Rotenone, an MRC complex I inhibitor was less e ective and azide, an MRC complex IV inhibitor, exhibited a marginal e ect. In contrast, antimycin A, an MRC complex III inhibitor, enhanced 4HPR-induced ROS generation. These ®ndings suggest that 4HPR enhances ROS generation by a ecting a target between complex II and complex III, presumably coenzyme Q. This e ect is followed by release of cytochrome c, increased caspase-3 activity, induction of MPT and eventual DNA fragmentation and cell death.

The adult cerebral cortex can adapt to environmental change. Using monocular deprivation as a paradigm, we find that rapid experience-dependent plasticity exists even in the mature primary visual cortex. However, adult cortical plasticity differs from developmental plasticity in two important ways. First, the effect of adult, but not juvenile monocular deprivation is strongly suppressed by administration of barbiturate just prior to recording visual evoked potentials, suggesting that the effect of adult experience can be inactivated acutely. Second, the effect of deprivation is less persistent over time in adults than in juveniles. This correlates with the known decline in CREB function during maturation of the visual cortex. To compensate for this decline in CREB function, we expressed persistently active VP16-CREB and find that it causes adult plasticity to become persistent. These results suggest that in development and adulthood, the regulation of a trans-synaptic signaling pathway controls the adaptive potential of cortical circuits.A primary function of the brain is to integrate the individual into a continually changing environment. Some aspects of this integration are accomplished through developmental processes, other aspects through learning. Although learning can occur throughout life, many behaviors, from language to sexual behavior, are shaped profoundly by early life experience. In this study, we have examined how the adaptive capacity of the cerebral cortex changes with maturation.A classical model of developmental plasticity is ocular dominance plasticity (Wiesel and Hubel 1963;Hubel and Wiesel 1998). Hubel and Wiesel showed that closing one eye of an infant cat produced a visual cortex dominated by the nondeprived eye. Closing an eye of an adult cat was ineffective. Single-unit studies in a number of mammalian systems, ranging from rodents to primates, have found that ocular dominance plasticity is restricted to a period prior to puberty (Hubel and Wiesel 1970;Blakemore et al. 1978;Olson and Freeman 1980;Issa et al. 1999;Fagiolini and Hensch 2000). The amount of deprivation required to alter the responses of visual cortical neurons depends on the animal's age. In the cat, during the peak of the critical period (4-5 wk of age), as little as 1 d of deprivation is sufficient to cause ocular dominance changes (Olson and Freeman 1975). Near the age of puberty, weeks or months of deprivation are necessary to induce changes observable by single-unit recordings, and the changes are thought to occur only in layers 2 and 3 of the visual cortex (Daw et al. 1992).In the clinical literature, however, there are reports suggesting that improvement of visual acuity can occur in adult patients with amblyopia, a central disorder of visual acuity, following patching of the normal eye (Selenow and Ciuffreda 1986; Saulles 1987; Rutstein and Fuhr 1992; Wick and Wingard 1992). Furthermore, a lengthy period of monocular occlusion caused by a dense cataract (Sloper and Collins 1995) can cause significant changes in visu...

Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic largedeletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNAdepleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Weekly cisplatin could be easier to manage than three-weekly cisplatin, because patients can be monitored more regularly for toxicity allowing the schedule to be altered if required. This regimen appears to be a suitable alternative to three-weekly high-dose cisplatin with concomitant radiotherapy.

The effect of fibroblast growth factor-2 (FGF-2) on synapse formation was investigated using rat cultured hippocampal neurons. Treatment with FGF-2 (0.4-10 ng/mL) for 6 days enhanced synaptogenesis on these neurons by approximately 50%, as determined by counting puncta immunostained for presynaptic- or postsynaptic-specific proteins. This enhancement was statistically significant, and was abolished by a specific inhibitor of mitogen-activated protein kinase (MAPK). The majority of neurons expressed FGF receptors (types 1-3) abundantly on the membrane of somata, dendrites, and growth cones, and in these regions phosphorylation of MAPK was enhanced after FGF-2 application. Furthermore, our experiments showed that the majority of synapses formed in cultures containing FGF-2 were positive both for presynaptic proteins and postsynaptic excitatory synapse-specific proteins, and that these synapses had a similar capacity to recycle the fluorescent styryl dye FM4-64 as those in the control culture. These results indicate that: (i) FGF-2 increases excitatory synapses on hippocampal neurons by activating MAPK activity through FGF receptors; and (ii) synapses formed in FGF-2-treated culture are capable of cycling vesicles.

Fluorescence spectroscopy has potential to improve cervical precancer detection. The relationship between tissue biochemistry and fluorescence is poorly understood. The goal of this study was to characterize normal cervical autofluorescence, using fresh tissue short-term tissue cultures and epithelial cell suspensions. Transverse, short-term tissue cultures were prepared from 31 cervical biopsies; autofluorescence images were obtained at 380 and 460 nm excitation. Fluorescence excitation-emission matrices were measured from normal, precancerous and cancerous cervical cell suspensions. Observed fluorescence patterns contrast those reported for frozen-thawed tissue, and were placed into groups with (1) bright epithelial and weak stromal fluorescence; (2) similar epithelial and stromal fluorescence; and (3) weak epithelial and bright stromal fluorescence. The average ages of women in the groups were 30.9, 38.0 and 49.2 years. Epithelial fluorescence intensity was similar in Groups 1 and 2, but weaker in Group 3. Stromal intensity was similar in Groups 2 and 3, but weaker in Group 1. The ratio of epithelial to stromal fluorescence intensity was significantly different for all groups. EEMs of cell suspensions showed peaks consistent with tryptophan, reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide. Short-term tissue cultures represent a novel, biologically appropriate model to understand cervical autofluorescence. Our results suggest a biological basis for the increased fluorescence seen in older, postmenopausal women.