Evaluation of a model for the effects of substrate interactions on the kinetics of reductive dehalogenation

The effects of primary electron-donor and electron-acceptor substrates on the kinetics of TCA biodegradation in sulfate-reducing and methanogenic biofilm reactors are presented. Of the common anaerobic electron-donor substrates that were tested, only formate stimulated the TCA biodegradation rate in both reactors. In the sulfate-reducing reactor, glucose also stimulated the reaction rate. The effects of formate and sulfate on TCA biodegradation kinetics were analyzed using a model for primary substrate effects on reductive dehalogenation. Although some differences between the model and the data are evident, the observed responses of the TCA degradation rate to formate and sulfate were consistent with the model. Formate stimulated the TCA degradation rate in both reactors over the entire range of TCA concentrations that were studied (from 50 g TCA/L to 100 mg TCA/L). The largest effects occurred at high TCA concentrations, where the dehalogenation kinetics were zero order. Sulfate inhibited the first-order TCA degradation rate in the sulfate-reducing reactor, but not in the methanogenic reactor. Molybdate, which is a selective inhibitor of sulfate reduction, stimulated the TCA removal rate in the sulfate-reducing reactor, but had no effect in the methanogenic reactor.     

Sustainable ecotourism in Costa Rica: the Monteverde Cloud Forest Preserve

The paper discusses the sustainability of the Monteverde Cloud Forest Preserve in the context of Costa Rican ecotourism. While the history of the Preserve is somewhat unique, the analysis of visitation, financial, ecological and economic factors provides a convincing case that tourism at the Preserve is sustainable. The experience of the Preserve is also put in the context of Costa Rican ecotourism, particularly to the national parks. The paper concludes that the Preserve has played a very important role in the development of Costa Rica as an ecotourism destination. Nonetheless, the failure of experience at the Preserve to inform recent changes in national park pricing policy reveal that Costa Rica has yet to fully capitalize on the experience gained and lessons learned at the Preserve.     

Evaluation of a remotely operated vehicle (ROV) as a tool for studying the distribution and abundance of zooplankton

Conventional methods for measuring zooplankton distributionsare too laborious and time consuming to permit sufficient temporaland spatial resolution in many instances. An ability to makemore efficient and precise measurements would be useful. Weevaluated the potential for using the video system of a commerciallyavailable remotely operated vehicle (ROV) to measure the distributionand abundance of zooplankton by calibrating ROV counts withcounts based on a conventional sampling procedure (a Schindlertrap), and by using an ROV to measure the density of zooplanktonin a small lake. As configured here, this particular ROV wassuitable for measuring the density of the cladocerans Daphniaand Holopedium. It was also suitable for assessing the distribution,but not absolute densities, of Chaoborus and Leptodora. Imagequality was inadequate for quantitative estimates of copepod(Diaptomus minutus) abundance, and prevented us from studyingbehavioral responses of copepods to the vehicle. We concludethat the ROV has at least three useful features: it can be usedto locate patches of those species that are imaged effectively;a large number of samples (videotapes) can be collected almostsynoptically with high spatial resolution; the ROV enables insitu observation of zooplankton. The ROV also has three importantlimitations: the small image volume makes it difficult to studyrare organisms; inadequate image resolution precludes studiesof relatively small organisms (e.g. the calanoid copepod D.minutus);zooplankton respond to the presence of the ROV.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the -glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R₂ pollen of R₁ progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin

Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.