Electrospray mass spectrometry of cis-[Ru(NO)Cl(bpy)2] (bpy=2,2-bipyridine): fragmentation from desolvated {[Ru(NO)Cl(bpy)2], Cl} ion pairs by electron transfer and internal Lewis base pathways

The electrospray mass spectrum (ESI-MS) of cis-[Ru(NO)Cl(bpy)₂]Cl₂ (bpy=2,2^′-bipyridine), obtained from 50% CH₃OH/50% H₂O as the mobile solvent, exhibited ruthenium-containing ions derived from a {[Ru^II(NO⁺)Cl(bpy)₂]²⁺, Cl⁻}⁺ ion pair (m/z=514) and [Ru^II(NO⁺)Cl(bpy)₂]²⁺ (m/z=239.5). [Ru^IIICl(bpy)₂]²⁺, from the loss of NO from the 239.5 ion, is detected at m/z=224.5. Only the m/z 514 ion pair is detected when 100% CH₃OH mobile solvent is used, but the presence of even small amounts of water prompted the additional detection of the m/z 239.5 and m/z 224.5 ions under tandem MS-MS conditions. Ruthenium-chloro-containing ions appear as a characteristic collection of eight main, and four lesser, intense ions created from combinations ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru, ⁹⁶Ru, ³⁵Cl and ³⁷Cl isotopes with minor contributions from ¹³C, etc. For convenience of discussion, only the most abundant m/z species are mentioned herein as representative of all the isotopically distributed ions.Four fragmentation channels are detectable from the m/z=514 chloride ion pair: (1) the loss of HCl (main channel; ca. 50% of fragmentation events), (2) the loss of NO (ca. 12% ), (3) the loss of bpy (minor pathway), and (4) the loss of Cl atom (ca. 38% ).Loss of NO from ion m/z 514 yields ion m/z 484, which is the precursor of ions m/z 448 (by loss of HCl), m/z 328 (by loss of bpy) and m/z 292 (by loss of HCl and bpy). Loss of HCl from ion m/z 514 generates ion m/z 478, [Ru^II(NO⁺)Cl(bpyH)(bpy-H)]⁺, deprotonated at the ortho C-H of one bpy ligand. In MS-MS experiments, the m/z 478 ion was established to undergo loss of NO, producing ion m/z 448, rejoining further fragmentation process for ion m/z 448 at this point. Loss of neutral bipyridine from m/z 514 in low yield produces ion m/z 358, which undergoes further loss of NO to form [RuCl₂(bpy)]⁺ ion (m/z=328). MS-MS “neutral loss of 30” spectra confirmed the NO loss events as part of the fragmentation sequence for all four pathways.A fourth species of m/z=479 from the “514” ion is obtained by an internal electron transfer from Cl⁻ of the ion pair, and loss of the resultant neutral Cl atom. The product [Ru^II(NO^·)Cl(bpy)₂]⁺ “479” fragment undergoes facile loss of NO to generate [Ru^IICl(bpy)₂]⁺ (m/z=449). Ion m/z 449 gives rise to ions m/z 413 (loss of HCl) and m/z 257(loss of HCl and bpy). MS-MS experiments confirm the neutral loss of Cl from the m/z 514 ion, and the formation of the m/z 449 ion via m/z 479 and m/z 514 parents. This pathway was not observed in a prior study for the related complex, [Ru(NO)Cl(dpaH)(dpa⁻)]⁺ (dpaH=2,2^′-dipyridylamine), which does not have an external Cl⁻ in an ion pair.     

The effect of an Escherichia coli regulatory mutation on transfer RNA structure.

The trpX mutation in Escherichia coli reduces trp operon attenuation in strains carrying wild-type tRNA^Trp. The trpX^? phenotype is alleviated (attenuation is restored) in UGA-suppressor tRNA^Trp-carrying strains (Yanofsky &; Soll, 1977).The tRNA from various trpX^? strains was characterized biochemically. Sequence analyses of wild-type tRNA^Trp and UGA suppressor tRNA^Trp, both derived from trpX^? strains, reveal an unmodified A in the position (adjacent to the anticodon) normally occupied by the hypermodified base ms²i⁶A.In addition, several tRNAs from trpX^? cells were characterized by RPC-5 column chromatography. We find that only tRNAs normally having ms²i⁶A exhibit altered elution profiles when compared to the homologous tRNAs from trpX^? cells. Introduction of the UGA suppressor into trpX^? cells does not restore normal Chromatographic behavior. These results suggest that the trpX gene product is necessary for the synthesis of ms²i⁶A. Thus, we propose that miaA (for the first gene involved in ms²i⁶A synthesis) replaces the trpX designation.The results reported here are discussed with regard to a model proposed by Lee &; Yanofsky (1977) in which efficient translation of the tandem trp codons in the leader sequence RNA is required for normal attenuation of the trp operon.     

Nutrient status of black spruce (Picea mariana [Mill.] BSP) forest soils dominated by Kalmia angustifolia L.

A study was conducted to determine soil chemistry in an uncut black spruce (Picea mariana) forest with and without the ericaceous understory shrub Kalmia angustifolia, as well as on a cut black spruce forest currently dominated by Kalmia. The organic (humus) and mineral (Ae, upper and lower B horizons) soils associated with Kalmia from uncut and cut forests, and non-Kalmia soils from uncut forest, were analyzed for selected soil properties. In general, mineral soils (B horizon) associated with Kalmia in uncut forest have lower values for organic matter (3.25%), organic nitrogen (0.66 mg·g⁻¹), Fe³⁺ (95.4 μg·g⁻¹) and Mn²⁺ (9 μg·g⁻¹), and higher values for pH (4.12) and Ca²⁺ (27 μg·g⁻¹) compared to non-Kalmia (organic matter, 3.43%; organic-N, 1.15 mg·g⁻¹; Fe³⁺, 431 μg·g⁻¹; Mn²⁺, 23.2 μg·g⁻¹; pH, 3.14; Ca²⁺, 15.6 μg·g⁻¹) and cut black spruce-Kalmia (organic matter, 3.74%; organic-N, 0.94 mg·g⁻¹; Fe³⁺, 379 μg·g⁻¹; Mn²⁺, 27 μg·g⁻¹; pH, 2.87; Ca²⁺, 25.2 μg·g⁻¹) forest. The high C:N ratio in Kalmia mineral soil from upper B (29.73) and lower B (identified as B+) (33.08) in uncut black spruce forest was recorded compared to non-Kalmia soils in B (18.17) and B+ (17.05) horizons in uncut black spruce forest. Phenolics leached out from Kalmia litter were lower in Kalmia associated soils than the non-Kalmia soils from the uncut forest, and Kalmia associated soils from the cut forest area. Results indicate that soils associated with Kalmia were nutrient poor particularly for nitrogen, phosphorus, iron and manganese, and provide some basis for the hypothesis that Kalmia has dominated microsites that were nutrient poor prior to Kalmia colonization.     

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG,FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX^G and FecX^B) and the third (FecG^H) in GDF9. All three mutations segregate in Belclare sheep while one, FecX^B, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX^G, FecX^B and FecG^H) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX^G mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX^B was only found among the hyper-prolific ewes. The FecG^H mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX^G and FecG^H mutations in Belclare and Cambridge sheep and that the FecX^B mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare.     

Use of near infrared reflectance spectroscopy (NIRS) for the prediction of the chemical composition and nutritional attributes of green crop cereals

The NIR spectra of 135 samples of the following green crop oats (Avena sativa), barley (Hordeum vulgare), triticale (x Triticosecale), wheat (Triticum durum), ryegrass (Lolium multiflorum) and sorghum (x Sorghum sudanense) grown in Portugal, were obtained by reflectance from oven-dried, milled whole plant material. Samples were cut at various stages of development (viz. stem elongation, boot, early heading, milk and dough stage) and were analysed by standard laboratory methods. The nutritive value was evaluated in vivo using rams in metabolic cages. The green crops showed values of ASH varying from 5.5 to 14.9%, crude protein (CP) from 5.0 to 22.3%, crude fibre (CF) from 25.3 to 40.4%, neutral detergent fibre (NDF) from 45.9 to 77.2%, acid detergent fibre (ADF) from 23.6 to 49.9%, acid detergent lignin (ADL) from 1.7 to 8.0%, dry matter digestibility (DMD) from 53.2 to 82.9%, organic matter digestibility (OMD) from 56.2 to 85.0% and dry matter intake (IVDMI) from 35.8 to 88.6 g DM kg⁻¹ W^0.75. Spectra were used to develop NIR calibration models for ASH (SEC=0.72; r²=0.90), CP (SEC=0.63; r²=0.98), CF (SEC=1.02; r²=0.94), NDF (SEC=1.41; r²=0.97), ADF (SEC=1.43; r²=0.96), ADL (SEC=0.49; r²=0.87) and in vivo attributes DMD (SEC=2.61; r²=0.86), OMD (SEC=2.36; r²=0.88) and IVDMI (SEC=6.05; r²=0.41). Calibration models were successful for all parameters except for IVDMI. However, animal attributes were less successfully predicted than chemical composition. Calibration models were generated omitting one of the six plant species to assess their ability to accommodate unknown green crop species not represented in the calibration model. In general, NIRS was shown to provide acceptable prediction of composition across different species of green crop cereals whether or not these were included in the calibration process.     

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na⁺/H⁺ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na⁺/H⁺ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na⁺/H⁺ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na⁺/H⁺ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na⁺/H⁺ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na⁺(Li⁺)/H⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na⁺(Li⁺)/H⁺ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na⁺/H⁺ antiporters. Also, the apparent K _m values of Hb_NhaD for Na⁺ and Li⁺ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na⁺/H⁺ antiporter.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

The desaturation and elongation of linolenic acid and eicosapentaenoic acid by hepatocytes and liver microsomes from rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil

The products of desaturation and elongation of [1−¹⁴C] 18:3(n − 3) and [1−¹⁴C]20:5(n − 3) were studied using hepatocytes and microsomes prepared from livers of trout maintained on diets containing either olive oil or fish oil, to establish the extent to which the formation of 22:6(n − 3) was enhanced in the absence of dietary 22:6(n − 3) and to investigate the pathway(s) of conversion of 18:3(n − 3) and 20:5(n − 3) to 22:6(n − 3). Levels of 20:5(n − 3) and 22:6(n − 3) in the total lipid of hepatocytes from trout fed olive oil were 20-fold and 10-fold, respectively, lower than in cells from trout fed fish oil. For both dietary groups, [1−¹⁴C]18:3(n − 3) was incorporated into hepatocyte lipid to a greater extent than [1−¹⁴C]20:5(n − 3). Almost 70% of the total radioactivity from [1−¹⁴C]18:3(n − 3) was recovered in hepatocyte triacylglycerols, whereas radioactivity from [1−¹⁴C]20:5(n − 3) was recovered almost equally in neutral lipids (52%) and polar lipids (48%). The products of desaturation and elongation from both labelled substrates were esterified mainly into hepatocyte polar lipids, whereas elongation products of [1−¹⁴C]18:3(n − 3) were preferentially incorporated into neutral lipids. Radioactivity recovered in the 22:6(n − 3) of polar lipids of hepatocytes from trout fed olive oil, from both ¹⁴C substrates, was approximately double that in hepatocytes from trout fed fish oil. No radioactivity from either [1−¹⁴C]18:3(n − 3) or [1−¹⁴C]20:5(n − 3) was incorporated into 22:6(n − 3) by microsomes isolated from livers from either group of fish and incubated in the presence of acetyl-CoA, malonyl-CoA, NADH, NADPH, ATP and coenzyme A. However, significant radioactivity was recovered in 24:5(n − 3) and 24:6(n − 3) from [1−¹⁴C]20:5(n − 3) and more radioactive 24:6(n − 3) accumulated in microsomes from trout fed olive oil than from trout fed fish oil. The results establish that the formation of 22:6(n − 3) from both 18:3(n − 3) and 20:5(n − 3) in hepatocytes of rainbow trout is stimulated by omitting 22:6(n − 3) from the diet and are consistent with the biosynthesis of 22:6(n − 3) in trout liver cells proceeding via 24:5(n − 3) and 24:6(n − 3) intermediates.     

Synthesis of selenocysteine by cysteine synthases from selenium accumulator and non-accumulator plants

Cysteine synthases were partially purified from leaf tissue of 3 selenium-accumulator species (Neptunia amplexicaulis, Astragalus racemosus and A. bisulcatus) and 4 non-accumulators (peas, white clover, A. sinicus and A. hamosus). The properties of all 7 enzymes with respect to cysteine synthesis from S^2? and O-acetylserine (OAS) were similar. All of the enzymes also catalysed the synthesis of selenocysteine when S^2? was replaced with Se^2?. There were no distinct differences between the properties of the enzymes from selenium-accumulator and non-accumulator plants with respect to selenocysteine synthesis. Se^2? inhibited the synthesis of cysteine and S^2? inhibited the synthesis of selenocysteine implying competition between S^2? and Se^2? for the enzyme. The affinities of the enzymes for Se^2? were substantially greater than for S^2?, and V_max (selenocysteine) was ca 7–48% of V_max (cysteine). Isolated pea chloroplasts catalysed the synthesis of selenocysteine from OAS and Se^2? at a rate of ca 22–26 μmol/mg Chl/hr. Sonicating the chloroplasts slightly enhanced the rate.     

Photobacterium malacitanum sp. nov., and Photobacterium andalusiense sp. nov., two new bacteria isolated from diseased farmed fish in Southern Spain

Three strains, H01100409B^T, H01100413B, and H27100402H^T, were isolated from several internal organs of diseased redbanded seabream (Pagrus auriga) reared in Andalusia (Southern Spain). All strains were studied by phenotypic, including chemotaxonomy, and genomic characteristics. Phylogenetic analysis based on concatenated sequences of six housekeeping genes (gyrB, ftsZ, topA, mreB, gapA, and 16S rRNA) supported the inclusion of the strains within the clade Phosphoreum of the genus Photobacterium, and two of the strains (H27100402H^T and H01100409B^T) formed a tight group separated from the closest species P. aquimaris. Genomic analyses, including average nucleotide identity (ANIb and ANIm) and DNA–DNA hybridization (DDH), clearly separated strains H27100402H^T and H01100409B^T from the other species within the clade Phosphoreum with values below the thresholds for species delineation. The chemotaxonomic features (including FAME analysis and MALDI-TOF-MS) of H27100402H^T and H01100409B^T strains confirmed their differentiation from the related taxa. The results demonstrated that strain H01100413B was classified as P. aquimaris and the strains H27100402H^T and H01100409B^T represented a new species each in the genus Photobacterium, for which we propose the names Photobacterium malacitanum sp. nov., type strain H27100402H^T (=CECT 9190^T = LMG 29992^T), and Photobacterium andalusiense sp. nov., type strain H01100409B^T (=CECT 9192^T = LMG 29994^T).     

Comparison between Biosynthesis of ent-Kaurene in Germinating Tomato Seeds and Cell Suspension Cultures of Tomato and Tobacco

Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures derived from tobacco callus (Nicotiana tabacum L.), tomato callus (Solanum lycopersicum L.), and in germinating tomato seeds. Incubation of extracts derived from the two cell cultures with either isopentenyl pyrophosphate-¹⁴C or with ¹⁴C-labeled mevalonate, followed by alkaline phosphatase hydrolysis, resulted in the formation of trans-geranylgeraniol-¹⁴C and trans-farnesol-¹⁴C. The corresponding pyrophosphates of trans-geranyl-geraniol-¹⁴C and trans-farnesol-¹⁴C were also detected. No detectable amount of ent-kaurene-¹⁴C was produced by these enzymatic preparations when trans-geranylgeranyl-¹⁴C pyrophosphate served as substrate. However, copalyl-¹⁴C pyrophosphate served as a substrate for the production of ent-kaurene. Cell-free extracts derived from germinating tomato seeds catalyzed the formation of ent-kaurene-¹⁴C from mevalonate-¹⁴C, isopentenyl-¹⁴C pyrophosphate, trans-geranylgeranyl-¹⁴C pyrophosphate, and copalyl-¹⁴C pyrophosphate.     

Actinoplanes hulinensis sp. nov., a novel actinomycete isolated from soybean root (Glycine max (L.) Merr)

A novel actinomycete, designated strain NEAU-M9^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-M9^T belonged to the genus Actinoplanes, being most closely related to Actinoplanes campanulatus DSM 43148^T (98.85 %), Actinoplanes capillaceus DSM 44859^T (98.70 %), Actinoplanes lobatus DSM 43150^T (98.30 %), Actinoplanes auranticolor DSM 43031^T (98.23 %) and Actinoplanes sichuanensis 03-723^T (98.06 %); similarity to other type strains of the genus Actinoplanes ranged from 95.87 to 97.56 %. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the isolate formed a distinct phyletic line with A. campanulatus DSM 43148^T and A. capillaceus DSM 44859^T. This branching pattern was also supported by the tree constructed with the maximum-likelihood method. However, the low level of DNA–DNA relatedness allowed the isolate to be differentiated from the above-mentioned two Actinoplanes species. Moreover, strain NEAU-M9^T could also be distinguished from the most closely related species by morphological, physiological and characteristics. Therefore, it is proposed that strain NEAU-M9^T represents a novel Actinoplanes species, Actinoplanes hulinensis sp. nov. The type strain of Actinoplanes hulinensis is NEAU-M9^T (= CGMCC 4.7036^T = DSM 45728^T).     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Permeation of a β-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS-(S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb³⁺-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val¹, Thr⁶)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg⁰, Trp⁵, Leu⁸)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

Radiation-induced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes

Whole blood cultures from humans and from the New World primate, Saguinus fuscicollis, were irradiated with various doses of 250 kV X-rays. The resulting centric ring plus dicentric aberration yields were fitted to the three models, Y = a+bD, Y = a+bD+cD², and Y = a+cD², by least squares regression. In both instances the best fit was to the model Y = a+bD+cD², with coefficients of the one- and two-track components for human and marmoset being: b = (0.78 ± 0.09)·10⁻³, c = (5.92 ± 0.31)·10⁻⁶, and b = (1.11 ± 0.36)·⁻³, c = (7.7 ± 1.7)·10⁻⁶, respectively.     

Electron paramagnetic resonance study of intersubunit interactions in nitrosyl-deoxy asymmetrical hybrid haemoglobin

The asymmetrical nitrosyl-deoxy hybrid haemoglobin, (α^NOβ^NO), (α^deoxyβ^deoxy), was prepared by removing oxygen with sodium dithionite from a mixture of oxyhaemoglobin and nitrosylhaemoglobin (Cassoly, 1978). This asymmetrical hybrid exhibited a distinctive triplet hyperfine structure in the electron paramagnetic resonance spectrum. This triplet has been shown to arise predominantly from the nitrosyl haem of an α subunit which has a deoxy-like structure (Nagai et al., 1978). By removing one or two carboxyl-terminal residues by carboxypeptidase digestion before mixing, one can obtain asymmetrical nitrosyl-deoxy hybrid haemoglobins in which only one of the four subunits is specifically modified. Eight such modified derivatives were examined by e.p.r.². They were (desArgα^NOβ^NO) (α^deoxyβ^deoxy), (desArg-Tyrα^NOβ^NO) (α^deoxyβ^deoxy), (α^NOdesHisβ^NO) (α^deoxyβ^deoxy), (α^NOdesHis-Tyr β^NO) (α^deoxyβ^deoxy), (α^NOβ^NO) (desArgα^deoxyβ^deoxy), (α^NOβ^NO) (desArg-Tyrα^deoxyβ^deoxy), (α^NOβ^NO) (α^deoxydesHisβ^deoxy) and (α^NOβ^NO) (α^deoxydesHis-Tyrβ^deoxy), where desArg, desArg-Tyr, desHis and desHis-Tyr indicate that the amino acids were removed from the carboxyl terminus of the subunit.The e.p.r. spectra for these eight derivatives have a more or less reduced relative intensity of the triplet, indicating that the non-covalent bonds involving carboxyl-terminal residues which stabilize the structure of deoxyhaemoglobin (Perutz, 1970) must all be intact in the unmodified asymmetrical nitrosyl-deoxy hybrid haemoglobin, (α^NOβ^NO) (α^deoxyβ^deoxy). By comparing the relative intensity of the triplet we were able to examine the effect of modification of one specific carboxyl terminus on the nitrosyl haem in the α₁ subunit. The effect was not symmetric, but increased in the order α₁ < β₂ < β₁ < α₁ (suffices 1 and 2 as defined by Perutz (1965)). We attribute this order to the non-equivalence of intersubunit interactions.     

Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.