A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.     

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.     

TX-1123: an antitumor 2-hydroxyarylidene-4-cyclopentene-1,3-dione as a protein tyrosine kinase inhibitor having low mitochondrial toxicity

A series of 2-hydroxyarylidene-4-cyclopentene-1,3-diones were designed, synthesized, and evaluated with respect to protein tyrosine kinase (PTK) inhibition, mitochondrial toxicity, and antitumor activity. Our results show that the cyclopentenedione-derived TX-1123 is a more potent antitumor tyrphostin and also shows lower mitochondrial toxicity than the malononitrile-derived AG17, a potent antitumor tyrphostin. The O-methylation product of TX-1123 (TX-1925) retained its tyrphostin-like properties, including mitochondrial toxicity and antitumor activities. However, the methylation product of AG17 (TX-1927) retained its tyrphostin-like antitumor activities, but lost its mitochondrial toxicity. Our comprehensive evaluation of these agents with respect to protein tyrosine kinase inhibition, mitochondrial inhibition, antitumor activity, and hepatotoxicity demonstrates that PTK inhibitors TX-1123 and TX-1925 are more promising candidates for antitumor agents than tyrphostin AG17.     

Difference in response by two cyprinid species to predatory threat from the nocturnal catfish Silurus asotus

Response to predators may not be identical between different prey species with different life histories and body sizes, particularly when the threat of predation is not great. To clarify this hypothesis, we introduced two prey species (10 Japanese dace, Tribolodon hakonensis, and 10 pale chub, Zacco platypus) into each experimental pond (in total, 8 ponds×4 trials) in which benthic algae had been allowed to grow. The presence or absence of Far Eastern catfish, Silurus asotus, and a refuge for prey fish was used to produce four treatments. The presence of catfish and/or a refuge did not affect either the feeding behavior or growth rate of Japanese dace. In contrast, when catfish were present and no refuge was available, the incidence of bottom feeding for pale chub greatly decreased. Pale chub growth rate was low when catfish were present and a refuge was available, indicating that pale chub spent more of their time in the refuge and lost opportunities of acquiring food. Japanese dace can reach a threshold size at which the prey are safe from predation, but pale chub cannot, and this may explain the differences in response to predators of the two species.     

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.