An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for -complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.     

G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E. coli host designed for assaying pZ189 supF mutational specificity.

Summary Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 × 10^–7 per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Specificity of recA441-mediated (tif-1) mutational events.

Summary To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI ^– mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G : C C : G, G : C T : A and A : T T : A transversion events were specifically enhanced after SOS induction. A preferential 5-Y-Purine-3 neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5-C/GTGG-3 sequences is also observed. Fifty events were recovered at the lacI frameshift hotspot site and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA⁺ background and is a consequence of the fourfold induction of the (–)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI ^– mutations.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 μg ml⁻¹, this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6 ± 2.8% live trophozoites remaining after 24 h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 μg ml⁻¹ of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 °C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 μg ml⁻¹ gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

Kinetics of ammonium, nitrate and phosphorus uptake by Canna indica and Schoenoplectus validus

Canna indica L. is an upright perennial rhizomatous herb, and Schoenoplectus validus (Vahl) A. Löve and D. Löve is a tall, perennial, herbaceous sedge. The nutrient uptake kinetics of C. indica and S. validus were investigated using the modified depletion method after plants were grown for 4 weeks in simulated secondary-treated wastewater. The maximum uptake rate (I_max) and Michaelis–Menten constant (K_m) were estimated by iterative curve fitting. The I_max for NH₄N (623 μmol g⁻¹ dry root weight h⁻¹) was significantly higher than that for NO₃N (338 μmol g⁻¹ dry root weight h⁻¹) in S. validus. In contrast, no difference was observed in C. indica. The I_max values for NO₃N and NH₄N were higher in S. validus than in C. indica. A significantly lower K_m was detected for NO₃N uptake in C. indica (385 μmol L⁻¹) compared to that in S. validus (1908 μmol L⁻¹). The I_max for PO₄P did not differ between the plant species. The K_m for PO₄P was significantly higher in C. indica (157 μmol L⁻¹) than in S. validus (60 μmol L⁻¹). In conclusion, we found that S. validus preferred NH₄N over NO₃N, had greater capacity for N uptake and higher affinity for PO₄P, but C. indica had greater affinity for NO₃N. Nutrient uptake capacity is likely related to habitat preference, and is influenced by the structure of roots and rhizomes.     

Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor

The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m⁻² day⁻¹ was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m⁻² day⁻¹. Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m⁻² day⁻¹) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m⁻² day⁻¹). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.