Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

Phylogenetics of Lophodermium from pine

Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.     

Chloride cell responses to long-term exposure to distilled and hard water in the gill of the armored catfish, Hypostomus tietensis (Loricariidae)

The morphological changes in the gill chloride cells of the armored catfish, Hypostomus tietensis , were investigated after 15 days' exposure to either distilled or hard water. The thickness of the water–blood barrier in the lamellae increased significantly in fish kept in distilled water due to the high proliferation of chloride cells. The apical surface of about 68% of chloride cells was sharply reduced by the development of an apical crypt with a sponge-like surface, although no change in the chloride cell fractional area was found. In contrast, H. tietensis kept in Na⁺, Cl⁻ and Ca²⁺ rich water displayed no significant changes in the number of chloride cells or in their apical surface morphology compared with the control fish. Chloride cell response to ion challenge in H. tietensis suggested the involvement of different strategies to maintain homeostasis in ion-poor water, which may be related to the life history of species.     

The influence of recovery duration on multiple sprint cycling performance

The purpose of this study was to examine the influence of recovery duration on various measures of multiple sprint cycling performance. Twenty-five physically active men completed 2 maximal multiple sprint (20 x 5 seconds) cycling tests with contrasting recovery periods (10 or 30 seconds). The mean +/- SD values for age, height, and body mass were 20.6 +/- 1.5 years, 177.2 +/- 5.4 cm, and 78.2 +/- 8.2 kg, respectively. All tests were conducted on a friction-braked cycle ergometer. Longer (30 seconds) recovery periods resulted in significantly (p < 0.05) higher measures of maximum (approximately 4%) and mean (approximately 26%) power output, the former appearing to result from a potentiation effect during the first few sprints. Thirty-second recovery periods also corresponded with significantly lower measures of fatigue (absolute difference: 16.1%; 95% likely range: 14.1-18.2%), heart rate, respiratory exchange ratio, and oxygen uptake. Blood lactate and ratings of perceived exertion (6-20 scale) increased progressively throughout both protocols and were significantly lower with 30-second recovery periods. The results of this study illustrate the considerable influence of recovery duration on various measures of multiple sprint work. Although the precise mechanisms of this response require further investigation, coaches and sport scientists should consider these findings when attempting to develop or evaluate the performance capabilities of athletes involved in multiple sprint sports.     

Vancomycin Resistance Among Strains of Staphylococcus epidermidis: Effects on Adherence to Silicone

Nosocomial device-related infections with Gram-positive cocci and their resistance to vancomycin are of increasing occurrence. We examined clinical isolates of relatively avirulent coagulase-negative staphylococci for their resistance to vancomycin and for their capabilities to adhere in vitro to medical grade silicone. Vancomycin resistance was found in 9 of 20 isolates, but there was no correlation between adherence capacity to silicone in the absence of vancomycin and vancomycin resistance for a given strain. Vancomycin in the medium, adsorbed to the surface of medical grade silicone or adsorbed on nongrowing cells, reduced adherence of representative Staphylococcus epidermidis to medical grade silicone. Received: 27 November 2000 / Accepted: 10 January 2001     

The atmospheric chemistry of trace gases and particulate matter emitted by different land uses in Borneo

We report measurements of atmospheric composition over a tropical rainforest and over a nearby oil palm plantation in Sabah, Borneo. The primary vegetation in each of the two landscapes emits very different amounts and kinds of volatile organic compounds (VOCs), resulting in distinctive VOC fingerprints in the atmospheric boundary layer for both landscapes. VOCs over the Borneo rainforest are dominated by isoprene and its oxidation products, with a significant additional contribution from monoterpenes. Rather than consuming the main atmospheric oxidant, OH, these high concentrations of VOCs appear to maintain OH, as has been observed previously over Amazonia. The boundary-layer characteristics and mixing ratios of VOCs observed over the Borneo rainforest are different to those measured previously over Amazonia. Compared with the Bornean rainforest, air over the oil palm plantation contains much more isoprene, monoterpenes are relatively less important, and the flower scent, estragole, is prominent. Concentrations of nitrogen oxides are greater above the agro-industrial oil palm landscape than over the rainforest, and this leads to changes in some secondary pollutant mixing ratios (but not, currently, differences in ozone). Secondary organic aerosol over both landscapes shows a significant contribution from isoprene. Primary biological aerosol dominates the super-micrometre aerosol over the rainforest and is likely to be sensitive to land-use change, since the fungal source of the bioaerosol is closely linked to above-ground biodiversity.     

Protective effects of acerola juice on genotoxicity induced by iron in vivo

Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron.     

Effect of Phosphorylation on EGFR Dimer Stability Probed by Single-Molecule Dynamics and FRET/FLIM

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (k_off). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the k_off of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.