Evidence for Iron-Dependent Nitrate Respiration in the Dissimilatory Iron-Reducing Bacterium Geobacter metallireducens

The dissimilatory iron-reducing bacterium Geobacter metallireducens was found to require iron at a concentration in excess of 50 μM for continuous cultivation on nitrate. Growth yield (~3-fold), cytochrome c content (~7-fold), and nitrate (~4.5-fold) and nitrite (~70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 μM iron.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.     

Zooplankton Growth,Respiration and Grazing on the Australian Margins of the Tropical Indian and Pacific Oceans

The specific activity of aminoacyl-tRNA synthetases (spAARS), an index of growth rate, and of the electron transport system (spETS), an index of respiration, was measured in three size fractions (73–150 μm, >150 μm and >350 μm) of zooplankton during five cruises to tropical coastal waters of the Kimberley coast (North West Australia) and four cruises to waters of the Great Barrier Reef (GBR; North East Australia). The N-specific biomass of plankton was 3–4-fold higher in the Kimberley than on the GBR in all 3 size classes: Kimberley 1.27, 3.63, 1.94 mg m^-3; GBR 0.36, 0.88 and 0.58 mg m^-3 in the 73–150 μm, >150 μm and >350 μm size classes, respectively. Similarly, spAARS activity in the Kimberley was greater than that of the GBR: 88.4, 132.2, and 147.6 nmol PPi hr^-1 mg protein ^-1 in the Kimberley compared with 71.7, 82.0 and 83.8 nmol PPi hr^-1 mg protein ^-1 in the GBR, for the 73–150 μm, >150 μm and >350 μm size classes, respectively. Specific ETS activity showed similar differences in scale between the two coasts: 184.6, 148.8 and 92.2 μL O₂ hr^-1 mg protein^-1 in the Kimberley, against 86.5, 88.3 and 71.3 μL O₂ hr^-1 mg protein^-1 in the GBR. On the basis of these measurements, we calculated that >150 μm zooplankton grazing accounted for 7% of primary production in the Kimberley and 8% in GBR waters. Area-specific respiration by >73 μm zooplankton was 7-fold higher in the Kimberley than on the GBR and production by >150 μm zooplankton was of the order of 278 mg C m^-2 d^-1 in the Kimberley and 42 mg C m^-2 d^-1 on the GBR. We hypothesize that the much stronger physical forcing on the North West shelf is the principal driver of higher rates in the west than in the east of the continent.     

Extracellular Polysaccharide Is Not Responsible for Aluminum Tolerance of Rhizobium leguminosarum bv. Phaseoli CIAT899

Strain UHM-5, a pSym^- Exo^- derivative of the aluminum-tolerant Rhizobium leguminosarum bv. phaseoli strain CIAT899, was equally tolerant of aluminum (Al) as the parental culture. Dialyzed culture supernatants of the wild-type cells grown in YEM broth (10⁹ cells ml^-1) contained 185 μg of glucose equivalents ml^-1 whereas UHM-5 culture supernatants yielded 2 μg of glucose ml^-1. The Exo^- derivative and the parental strain gave essentially similar growth in medium containing from 0 to 300 μM Al, indicating that the pSym of CIAT899, and extracellular polysaccharide, were not involved in the aluminum tolerance of this strain. However, increasing the level of Al from 80 to 150 μM increased the lag phase, induced a slight killing of the inoculum, and depressed the final populations by about fivefold. Doubling the aluminum concentration from 150 to 300 μM presented a severe aluminum stress to CIAT899 and UHM-5: the inoculum level dropped 10-fold, indicating killing of the inoculum, and remained depressed for ca. 4 days before continuing to grow slowly; the final population was decreased 15-fold relative to that of cultures grown in medium containing 80 μM Al. The production by CIAT899 of other extracellular or intracellular aluminum tolerance factors was investigated in culture by using aluminum-sensitive rhizobia as stress indicators. These experiments, conducted at 80 μM Al, demonstrated that CIAT899 produced neither extracellular nor intracellular products that could alleviate toxicity for the Al-sensitive indicator rhizobia.     

A Xanthine-Derivative K+-Channel Opener Protects against Serotonin-Induced Cardiomyocyte Hypertrophy via the Modulation of Protein Kinases

This study investigated whether KMUP-1, a xanthine-derivative K⁺ channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca²⁺ channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 μM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 μM), and attenuated by the LTCC blocker verapamil (1 μM) and the 5-HT_2A antagonist ketanserin (0.1 μM), but unaffected by the 5-HT_2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca²⁺ currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (I_Ca,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 μM). Serotonin-increased I_Ca,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 μM and chelerythrine, 1 μM) while the current was enhanced by the PKC activator PMA, (1 μM) but not the PKA activator 8-Br-cAMP (100 μM), and was abolished by KMUP-1. In contrast, serotonin-increased I_Ca,L was blunted by the PKG activator 8-Br-cGMP (100 μM), but unaffected by the PKG inhibitor KT5823 (1 μM). Notably, KMUP-1 blocked serotonin-increased I_Ca,L but this was partially reversed by KT5823. In conclusion, serotonin-increased I_Ca,L could be due to activated 5-HT_2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.     

Sorption and Release of Organics by Primary,Anaerobic, and Aerobic Activated Sludge Mixed with Raw Municipal Wastewater

New activated sludge processes that utilize sorption as a major mechanism for organics removal are being developed to maximize energy recovery from wastewater organics, or as enhanced primary treatment technologies. To model and optimize sorption-based activated sludge processes, further knowledge about sorption of organics onto sludge is needed. This study compared primary-, anaerobic-, and aerobic activated sludge as sorbents, determined sorption capacity and kinetics, and investigated some characteristics of the organics being sorbed. Batch sorption assays were carried out without aeration at a mixing velocity of 200 rpm. Only aerobic activated sludge showed net sorption of organics. Sorption of dissolved organics occurred by a near-instantaneous sorption event followed by a slower process that obeyed 1^st order kinetics. Sorption of particulates also followed 1^st order kinetics but there was no instantaneous sorption event; instead there was a release of particles upon mixing. The 5-min sorption capacity of activated sludge was 6.5±10.8 mg total organic carbon (TOC) per g volatile suspend solids (VSS) for particulate organics and 5.0±4.7 mgTOC/gVSS for dissolved organics. The observed instantaneous sorption appeared to be mainly due to organics larger than 20 kDa in size being sorbed, although molecules with a size of about 200 Da with strong UV absorbance at 215–230 nm were also rapidly removed.     

A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-β and Regulates Its Clearance from the Mouse Brain

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ∼70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw^+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw^+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy.     

A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter⁻¹) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter⁻¹. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of Pseudomonas fluorescens P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO₄-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO₄-P liter⁻¹. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter⁻¹ (median, 0.60 μg of P liter⁻¹).     

Endothelial Surface Glycocalyx Can Regulate Flow-Induced Nitric Oxide Production in Microvessels In Vivo

Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA). Rats (SD, 250–300g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles

Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low-light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum × morifolium Ramat. “Fiesta” and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (μmol m⁻²s⁻¹). The CO₂ assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO₂ assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO₂ assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 μmol m⁻² s⁻¹ PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 μmol m⁻² s⁻¹, high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 μmol m⁻² s⁻¹), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 μmol m⁻² s⁻¹) or mean irradiance (200-300-200 μmol m⁻² s⁻¹).     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.     

Technique for Simultaneous Determination of [35S]Sulfide and [14C]Carbon Dioxide in Anaerobic Aqueous Samples

A technique for the simultaneous determination of [³⁵S]sulfide and [¹⁴C]carbon dioxide produced in anaerobic aqueous samples dual-labeled with [³⁵S]sulfate and a ¹⁴C-organic substrate is described. The method involves the passive distillation of sulfide and carbon dioxide from an acidified water sample and their subsequent separation by selective chemical absorption. The recovery of sulfide was 93% for amounts ranging from 0.35 to 50 μmol; recovery of carbon dioxide was 99% in amounts up to 20 μmol. Within these delineated ranges of total sulfide and carbon dioxide, 1 nmol of [³⁵S]sulfide and 7.5 nmol of [¹⁴C]carbon dioxide were separated and quantified. Correction factors were formulated for low levels of radioisotopic cross-contamination by sulfide, carbon dioxide, and volatile organic acids. The overall standard error of the method was ±4% for sulfide and ±6% for carbon dioxide.     

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min⁻¹. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 μM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 μM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 μg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 μg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Cocultivation of avian orthomyxoviruses and paramyxoviruses in embryonated eggs: implications for surveillance studies.

A relatively inexpensive flow microcalorimeter is described which is capable of detecting heat outputs as low as 3 μW (precision, ±2%). Its use is illustrated on river epilithon (0.8 to 6.8 μW cm⁻²), river sand (9.8 μW cm⁻³), and marine sand (15.3 μW cm⁻³); however, it could be used to detect the heat output from any biotic material over which a flow of water can be passed, provided that such an action would not be disruptive to chemical and biological equilibria.     

Giant Clams and Rising CO2: Light May Ameliorate Effects of Ocean Acidification on a Solar-Powered Animal

Global climate change and ocean acidification pose a serious threat to marine life. Marine invertebrates are particularly susceptible to ocean acidification, especially highly calcareous taxa such as molluscs, echinoderms and corals. The largest of all bivalve molluscs, giant clams, are already threatened by a variety of local pressures, including overharvesting, and are in decline worldwide. Several giant clam species are listed as ‘Vulnerable’ on the IUCN Red List of Threatened Species and now climate change and ocean acidification pose an additional threat to their conservation. Unlike most other molluscs, giant clams are ‘solar-powered’ animals containing photosynthetic algal symbionts suggesting that light could influence the effects of ocean acidification on these vulnerable animals. In this study, juvenile fluted giant clams Tridacna squamosa were exposed to three levels of carbon dioxide (CO₂) (control ~400, mid ~650 and high ~950 μatm) and light (photosynthetically active radiation 35, 65 and 304 μmol photons m^-2 s^-1). Elevated CO₂ projected for the end of this century (~650 and ~950 μatm) reduced giant clam survival and growth at mid-light levels. However, effects of CO₂ on survival were absent at high-light, with 100% survival across all CO₂ levels. Effects of CO₂ on growth of surviving clams were lessened, but not removed, at high-light levels. Shell growth and total animal mass gain were still reduced at high-CO₂. This study demonstrates the potential for light to alleviate effects of ocean acidification on survival and growth in a threatened calcareous marine invertebrate. Managing water quality (e.g. turbidity and sedimentation) in coastal areas to maintain water clarity may help ameliorate some negative effects of ocean acidification on giant clams and potentially other solar-powered calcifiers, such as hard corals.