Anti-invasive effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), a natural inhibitor of metallo and serine proteases

Several reports have attributed to green tea chemopreventive and therapeutic properties. Epidemiological studies have linked the regular use of green tea to a reduced incidence of breast and colon carcinomas. Tea contains several antioxidants, including polyphenols of the catechin (green tea) and theaflavin (black tea) groups. Green tea derivatives have been shown to act in vitro and in vivo as anti-inflammatory, anti-viral and anti-tumor drugs. Despite the extensive body of data only few studies have investigated the molecular mechanisms underlying these effects. In this brief review we focus on the inhibitory activity of catechins derived from green tea toward proteases involved in tumor invasion.     

An interface model for the periodontal ligament

A nonlinear interface constitutive law is formulated for modeling the mechanical behavior of the periodontal ligament. This gives an accurate interpolation of the few available experimental results and provides a reasonably simple model for mechanical applications. The model is analyzed from the viewpoints of both mathematical consistency and effectiveness in numerical calculations. In order to demonstrate the latter, suitable two- and three-dimensional nonlinear interface finite elements have been implemented.     

A continuous kinetic assay for RNA-cleaving deoxyribozymes,exploiting ethidium bromide as an extrinsic fluorescent probe

We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.     

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.     

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.     

Conidial anastomosis tubes in Colletotrichum

We describe the occurrence of special kinds of hyphae that create anastomoses directly between conidia. They can be found both in the laboratory and on infected plants. They first appear within asexual fruiting bodies approximately 15 days after conidiation has begun leading to the appearance of chains of connected conidia. Coincident with this we demonstrate in Colletotrichum lindemuthianum nuclear dynamics, including fragmentation, with cytoplasmic flow and passage of nuclei and organelles between conidia through the anastomosis tubes. We propose that conidial anastomosis tubes play an important role in the life cycle of these fungi.     

Platinum toxicity and gene expression in Xenopus embryos: analysis by FETAX and differential display

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay-Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen alpha-chain and a novel gene were identified.     

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans

Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561-575 nm, 552-540 nm, and 602-630 nm indicated the presence of three b-type cytochromes (Em,7 of +244 +/- 8, +24 +/- 3, -163 +/- 11 mV), four c-type cytochromes (Em,7 of +280 +/- 10, +210 +/- 5, +125 +/- 8, and 20 +/- 3 mV) and two a-type cytochromes (Em,7 of +335 +/- 15, +218 +/- 18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50 = 2 microM) and azide (I50 = 1 mM), while a soluble cytochrome c (c551, Em,7 = +217 +/- 2 mV) was shown to be the physiological electron carrier connecting the bc1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh > or = +80 < or = +140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.     

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3. © Chapman & Hall     

Seasonal succession of Cladocerans in a ricefield in Italy

A biological ricefield in northern Italy, without periodic dry spells in its growing cycle and therefore more familiar to naturally humid zones, was studied for its heleoplankton community. The biocoenosis reached a greater level of complexity than reported in literature. In particular, the seasonal succession of Cladocerans, the dominant group throughout the study period, was analyzed. Wlassicsia pannonica (Daday, 1904; Anomopoda Macrothricidae), is new to Italy; its morphology is compared to that of other populations of the same species and its biological cycle is compared with that of other dominant Cladocerans.