Cloning (CAG/GTC)n STSs by an Alu-(CAG/GTC)n PCR method: an approach to human chromosome 12 and spinocerebellar ataxia 2 (SCA2).

We report here an Alu-(CAG/GTC)n PCR method for the cloning of STSs with (CAG/GTC)n sequences. We have applied this method to genomic DNA of a somatic cell hybrid containing human chromosome 12 where linkage has been found for a known familiar dominant ataxia (SCA2), which is thought to be due to a (CAG/GTC)n expansion. We have isolated several clones containing (CAG/GTC)n sequences, which include previously identified sequences that map to chromosome 12. This method could be a new PCR approach for the cloning of repeats based on their proximity to Alu sequences.     

Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

Bacterial infection of mudfish Clarias gariepinus (Siluriformes: Clariidae) fingerlings in tropical nursery ponds

Bacterial infection among the most common cultured mudfish Clarias gariepinus in Africa, has become a cause of concern, because it constitutes the largest economic loss in fish farms. In order to provide useful biological data of the pathogens for good management practices, samples were collected monthly between January 2008 and December 2009 in three monoculture nursery ponds, located in three different positions: upriver (A, grassland), mid-river (B, mixed forest and grassland) and downriver (C, rainforest) along 200 km length of Cross River floodplains, Nigeria. A total of 720 fingerlings between 15.1 and 20.7 g were analyzed to determine the degree of infection. The bacterial pathogens were taken from their external surfaces, and were isolated and identified by standard methods. The caudal fins of fingerlings from pond A had the highest bacterial load (5.8 x 10(3) cfu/g), while the least counts (1.2 x 103 cfu/g) were identified on the head of fish from pond C, with Flexibacter columnaris as the major etiological agent. Pseudomonas fluorescens, Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus and Micrococcus luteus were identified as co-isolates with P. fluorescens as dominant (0.7 x 10(2) cfu/mL) co-isolates in pond water. Clinical signs of five white spots with red periphery appeared on the external surface of infected fish. All the fish sampled, died after 4 to 9 days. There was no significant difference in the bacterial counts between different ponds, but the difference between fish organs/parts examined was significant. Fish from these ponds are therefore potentially dangerous to consumers and highly devalued, with the economic impact to producers. Preventive methods to avoid these infections are recommended.     

Denitrification and methane production in sediment of Hamilton Harbour (Canada)

Systematic sampling of 21 sites covering Hamilton Harbour (Lake Ontario, Canada) was carried out during the summer in 1990 and 1991 in order to study how well environmental factors, such as O₂, NO ₃ ^– , and organic carbon, and the spatial structure can explain observed variation of potential denitrification, CH₄ and CO₂ production, as well as N₂ fixation in sediment slurries. Using canonical redundancy analysis and an extension of this method to partial out the variance into spatial and environmental components, we found that most of the explained fraction of potential microbial activities (70–90%) was accounted for by the significant environmental variables (NH ₄ ⁺ , particulate carbon, dissolved organic carbon, dissolved O₂, depth, and temperature) and not much by the spatial polynomial trend surface. We found significant path coefficients (0.53 and 0.57 in 1990 and 1991) between CO₂ production and potential denitrification, which suggests that denitrifiers are dependent upon a heterotrophic bacterial population for directly assimilable carbon sources. We also found significant path coefficients between particulate carbon and both CH₄ production (0.67 and 0.33) and CO₂ production (0.50 and 0.38), while significant path coefficients were also found between dissolved organic carbon and CO₂ production (0.34 and 0.47). We conclude that beside well-known abiotic factors such as O₂, NO ₃ ^– , and organic carbon, a biotic factor involved in carbon metabolism may be important in explaining the spatial variation of denitrification capacity in the sediment of Hamilton Harbour.Correspondence to: R. Roy.     

Development of the stable isotope tracer approach for studies of copper turnover in the rat and mouse

The stable isotope tracer approach was explored for long-term investigations of copper turnover in the adult rat and mouse, with inductively coupled plasma mass spectrometry for isotope measurements. The isotopic measurement method permitted precision and accuracy of <1.0%, with an overall sample blank of <0.05 microg copper. Rats were fed a copper-deficient diet and deionized water with (+Cu) or without (-Cu) copper (20 microg/ml). Both groups underwent a single-day replacement of drinking water with 20 microg/ml of (65)Cu. Compared with the baseline isotope ratio ((65)Cu/(63)Cu) of 0.462 +/- 0.002, blood plasma ratios for the +Cu group on days 2, 7, and 14 postdosing were 0.702 +/- 0.021, 0.557 +/- 0.004, and 0.474 +/- 0.001, respectively. The corresponding data for liver were 1.652 +/- 0.018, 0.560 +/- 0.005, and 0.482 +/- 0.001, respectively. For the -Cu group, respective plasma ratios were 1.580 +/- 0.04. 0.917 +/- 0.02, and 0.664 +/- 0.01 for days 2, 7, and 14 postdosing, and the ratios for liver were 0.987 +/- 0.02, 0.876 +/- 0.04, and 0.739 +/- 0.03. Mice previously made copper deficient to varying degrees were given a single-day replacement with the label. When the 24-hour postdosing isotope ratios in the livers of these mice were correlated with the activity of plasma ceruloplasmin, a negative correlation (r = -0.85) was observed. Isotope enrichment in both rats and mice was greater in the copper-deficient animals compared with the controls.     

Planktonic bacteria in white shrimp (Litopenaeus vannamei) and channel catfish (Letalurus punetaus) aquaculture ponds in a salt-alkaline region

Aquaculture in salt-alkaline regions is encouraged in China, and culture of many aquatic species has been introduced into these areas. In this study, we cultured two species, white shrimp (Litopenaeus vannamei) and channel catfish (Letalurus punetaus) separately in aquaculture ponds in a salt-alkaline region in northwest China and assessed the impacts of the aquaculture operations on the planktonic bacterial community in the culture ponds. Culture of both species decreased the planktonic bacterial diversity and altered the bacterial community structure in the aquaculture ponds compared with the source water. Among the 10 dominant bacterial phyla, 8 were significantly correlated with environmental parameters; the exception was Actinobacteriota, the most dominant phylum, and Firmicutes. Proteobacteria and Bacteroidota abundances showed significant positive correlations with alkalinity, whereas Patescibacteria, Cyanobacteria, Planctomycetota, and Verrucomicrobiota abundance were positively correlated with salinity. Linear regression analysis showed that alkalinity was positively correlated with bacterial beta diversity and salinity was negatively correlated with that. In addition, white shrimp aquaculture significantly lowered the alkalinity, which suggests that culture of this species in inland salt-alkaline regions is a potential dealkalization solution.     

(99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of (99m)Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z(HER2:342) with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of (99m)Tc-maGEG-Z(HER2:342) and (99m)Tc-maEEE-Z(HER2:342) was compared with (99m)Tc-maGGG-Z(HER2:342) in normal mice, and the tumor targeting properties of (99m)Tc-maEEE-Z(HER2:342) were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for (99m)Tc-labeling of Z(HER2:342) reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. (99m)Tc-maEEE-Z(HER2:342) showed a receptor-specific tumor uptake of 7.9 +/- 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with (99m)Tc-maEEE-Z(HER2:342) could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for (99m)Tc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making (99m)Tc-maEEE-Z(HER2:342) a promising tracer for clinical imaging of HER2 overexpression in tumors.     

Use of an organoleptic tracer (mercaptan) for testing for leaks in safety equipment

A safe, rapid, sensitive, and economical method is presented for detecting leaks in gas-tight laboratory cabinets and other apparatus. This method, using no fluorocarbons and requiring no prior decontamination, is effective under negative, neutral, or positive chamber conditions.     

Resin effects in solid phase S(n)Ar and S(n)2 macrocyclizations

Seven different supports were compared in solid-phase S(N)Ar and S(N)2 macrocyclization reactions. Product purities were assayed for a relatively facile ring-closure process to give products 1 and 3. Some less-facile ring-closure reactions give the undesired dimeric macrocyclization by-products 2; some of these more-demanding ring closures were also examined. Finally, experiments were performed to gauge the rate of cyclizations on different resins, and some qualitative data were obtained for this.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H(+) leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H(+) leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenal-induced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed.     

Use of an organoleptic tracer (mercaptan) for testing for leaks in safety equipment.

A safe, rapid, sensitive, and economical method is presented for detecting leaks in gas-tight laboratory cabinets and other apparatus. This method, using no fluorocarbons and requiring no prior decontamination, is effective under negative, neutral, or positive chamber conditions.     

The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.

     

N Kinetic Analysis of N(2)O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the "denitrifying enzyme" nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% N) and 0.4 mM nitrite (99% N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.     

Sulphur accumulation and redistribution in wheat (Triticum aestivum): a study using stable sulphur isotope ratios as a tracer system

Wheat plants were grown hydroponically and fed with two sulphate sources differing in stable isotope composition, one having a δ ³⁴S of 13·7‰ and the other 4·1‰. Plant sulphur (S) isotope ratios were determined using an on-line continuous flow-isotope ratio mass spectrometer. This method greatly simplified the procedure for the measurement of S isotope ratios, and was found to be precise for samples containing > 1 mg S g^–1 dry weight. The δ ³⁴S values of plant shoots, which had been grown on a single sulphate source, were very close to the source values, suggesting little isotope fractionation during sulphate uptake and transport from roots to shoots. By changing the sulphate sources at different growth stages, it was possible to estimate S accumulation and redistribution within different plant parts. At maturity, wheat grain derived 14, 30, 6 and 50% of its S from the accumulation during the following successive growth stages: between emergence and early stem extension, between stem extension and flag leaf emergence, between flag leaf emergence and anthesis, and after anthesis, respectively. It was estimated that 39, 32 and 52% of the S present in the flag leaves, older leaves and stems, respectively, at anthesis, was exported during the postanthesis period. These results demonstrate considerable cycling of S within wheat plants, and highlight the importance of S uptake after anthesis to the accumulation of S in grain under the experimental conditions employed.     

Biotic interactions in Lake Xolotlán (Managua): An integrating approach

An integral view of the trophic aspects so far obtained from studies of phytoplankton, zooplankton, benthos and fish in Lake Xolotlán (Managua) is presented as a first contribution to the analysis of the lake ecosystem.     

Potential use of ageing cultures ofIsochrysis aff.galbana (Isochrysis Tahitian,T. Iso) as starter cultures for live algal food production in tropical aquaculture

When grown in batch culture for a prolonged period (up to 11 months), the cells ofIsochrysis aff.galbana (Isochrysis Tahitian strain T. Iso) lost their capacity to divide and became enlarged, with a reduction in the content of photosynthetic pigment and photosynthetic capacity. Upon returning to fresh medium, the cells of a 11-month-old culture regained their capacity to divide within 3 days under light conditions. The resulting cells possessed similar size, photosynthetic pigment and photosynthetic capacity as those of logarithmic growth phase. The results suggest that the aged cultures ofIsochrysis can be used as starter cultures for the production of live food for aquaculture animals.