Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Edible oil degradation by using yeast coculture of <Emphasis Type="Italic">Rhodotorula pacifica</Emphasis> ST3411 and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> ST3412

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Antibacterial Activities of Novel Diselenide-bridged <Emphasis Type="Italic">bis</Emphasis>(Porphyrin)s on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Investigated by Microcalorimetry

The actions of two novel diselenide-bridged bis(porphyrin)s (1 and 2) on Staphylococcus aureus growth was investigated by microcalorimetry at 37.00°C, compared with that of Na₂SeO₃. Differences in their capacities to inhibit the growth metabolism of S. aureus were observed. By analyzing the power–time curves, crucial parameters such as the rate constant of bacterial growth (k), inhibitory rate (I), and generation time (t _G) were determined. The growth rate constant (k) of S. aureus (in the log phase) in the presence of the drugs decreased with increasing concentrations of the drugs regularly. The relationship of k and c is nearly linear for diselenide-bridged bis(porphyrin) 2. The sequence of the antibacterial activities of these selenium compounds tested was 2 > 1 > Na₂SeO₃.     

Valsartan Orodispersible Tablets: Formulation, <Emphasis Type="Italic">In vitro</Emphasis>/<Emphasis Type="Italic">In vivo</Emphasis> Characterization

Valsartan orodispersible tablets have been developed at 40-mg dose, with the intention of facilitating administration to patients experiencing problems with swallowing and hopefully, improving its poor oral bioavailability. Work started with selecting drug compatible excipients depending on differential scanning calorimetric analysis. A 3³ full factorial design was adopted for the optimization of the tablets prepared by freeze-drying technique. The effects of the filler type, the binder type, and the binder concentration were studied. The different tablet formulas were characterized for their physical properties, weight variation, disintegration time, surface properties, wetting properties, and in vitro dissolution. Amongst the prepared 27 tablet formulas, formula number 6 (consisting of 4:6 valsartan:mannitol and 2% pectin) was selected to be tested in vivo. Oral bioavailability of two 40 mg valsartan orodispersible tablets was compared to the conventional commercial tablets after administration of a single dose to four healthy volunteers. Valsartan was monitored in plasma by high-performance liquid chromatography. The apparent rate of absorption of valsartan from the prepared tablets (C _max = 2.879 μg/ml, t _max = 1.08 h) was significantly higher than that of the conventional tablets (C _max = 1.471 μg/ml, t _max = 2.17 h), P ≤ 0.05. The relative bioavailability calculated as the ratio of mean total area under the plasma concentration–time curve for the orodispersible tablets relative to the conventional ones was 135%. The results of the in vivo study revealed that valsartan orodispersible tablets would be advantageous with regards to improved patient compliance, rapid onset of action, and increase in bioavailability.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

Bioactive metabolites from <Emphasis Type="Italic">Phoma</Emphasis> species,an endophytic fungus from the Chinese medicinal plant <Emphasis Type="Italic">Arisaema erubescens</Emphasis>

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC₅₀ values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC₅₀ values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.     

Quantitative Structure-Activity Relationships for the Pre-Steady State of <Emphasis Type="Italic">Pseudomonas Species</Emphasis> Lipase Inhibitions by <Emphasis Type="Italic">p</Emphasis>-Nirophenyl-<Emphasis Type="Italic">N</Emphasis>-Substituted Carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, K_S, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k₂ are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k_obs) versus inhibition concentration ([I]) plot against k_obs=k₂+k₂[I]/(K_S+[I]). Values of pK_S, and log k₂ are linearly correlated with the σ^* values with the ρ^* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the ρ^* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the ρ^* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

Molecular characterization,recombinant expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and biological activity of (<Emphasis Type="Italic">S</Emphasis>)-Tetrahydroberberine oxidase from <Emphasis Type="Italic">Corydalis saxicola</Emphasis> Bunt

(S)-Tetrahydroberberine [(S)-THB] oxidase is the last enzyme of benzylisoquinoline alkaloids pathway which catalyzes the dehydrogenation of four hydrogen atoms of (S)-THB to produce berberine, the final step of berberine biosynthesis. A (S)-THB gene, designated as Cs(S)-THBO (Genbank accession No. HQ393909), was cloned from a Corydalis saxicola cDNA library by rapid amplification of cDNA ends. The full-length of cDNA of Cs(S)-THBO was 1127 bp with an open reading frame of 699 bp that predicted to encode a 232-amino acid polypeptide, with a predicted molecular mass of 25.20 kDa. Cs(S)-THBO was the first (S)-THBO gene found in C. saxicola. Real-time quantitative PCR analysis indicated that Cs(S)-THBO was constitutively expressed in roots, stems, leaves and flowers of C. saxicola, and with the highest expression level in roots. The results of treatment experiment for plant defense responses revealed that expression of Cs(S)-THBO had a prominent diversity. Recombinant Cs(S)-THBO protein expressed in Escherichia coli strain BL21 (DE3) was active. The results of feeding experiment and HPLC–DAD–ESI–MSⁿ analysis showed that Cs(S)-THBO had the function of catalyzing (S)-tetrahydroberberine to berberine.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

The nodule conductance to O<Subscript>2</Subscript> diffusion increases with high phosphorus content in the <Emphasis Type="Italic">Phaseolus vulgaris-</Emphasis>rhizobia symbiosis

Although recent studies have addressed the effects of phosphorus (P) deficiency on nodule O₂ permeability, little attention has been given to the relationship between nodule P status and nodule permeability. To study these traits, four recombinant inbred lines, namely RILs 34, 83, 115, 147 and one local variety (Concesa) of common bean (Phaseolus vulgaris) were inoculated with RhM11 (a native rhizobial strain from Haouz area of Marrakesh), and grown in hydroaeroponic culture under P-sufficiency (250 μmol P plant⁻¹ week⁻¹) versus P-deficiency (75 μmol P plant⁻¹ week⁻¹) conditions. At the flowering stage, the biomass of plants and nodules and their P contents was determined after measuring O₂ uptake by nodulated roots (Conr) and nodule conductance to O₂ diffusion (g_n). The results showed that P-deficiency significantly decreased plant growth and nodulation, though there were differences between bean genotypes. P-deficiency also induced a decrease in nodule P content (31%) in both sensitive (83, 147) and tolerant lines (34, 115), a 42 and 27% reduction in shoots of sensitive and tolerant lines, respectively. These decreases were associated with significant variations in nodule surface and O₂ permeability among bean genotypes and P-nutrition. Under P-deficiency, g_n increased more for the sensitive (39%) than for the tolerant lines (27%). This increase was linked with a rise both in the P levels in nodules and shoots, as well as in the efficiency of symbiotic nitrogen fixation as determined by nodule-dependent biomass production for the sensitive lines. Furthermore, positive correlations were found between O₂ permeability, g_n and P content both in nodules and shoots (r ² = 0.94** and r ² = 0.96**). We conclude that nodule variations in Conr and g_n are related to nodule P content, and may contribute to the adaptation of energy metabolism in N₂-fixing bean nodules to P-deficiency.     

The functional <Emphasis Type="Italic">SLC11A1</Emphasis> gene polymorphisms are associated with sarcoidosis in Turkish population

Sarcoidosis (SA) is an immune-mediated multisystemic disorder of unknown etiology characterized by the accumulation of lymphocytes, mononuclear phagocytes and epithelioid cell granulomas involved in different organs and tissues. The belief that genetics contribute to SA etiology is supported by twin studies, disease clustering in families and racial differences in incidence rates. Involvements of SLC11A1 in macrophage function and activation, makes it an attractive candidate gene for immune-mediated and infectious diseases. We investigated the association between SA and four polymorphisms of the SLC11A1 gene, including a single nucleotide change in intron 4 (INT4); a nonconservative single-base substitution at codon 543 (D543N); a TGTG deletion in the 3′ untranslated region; and the functional (GT)_n repeat polymorphism in the 5′ region, in 95 Turkish SA patients and 150 healthy controls, by using amplification refractory mutation system–polymerase chain reaction and sequencing. We found significant association between SA and INT4 G/C allele frequency (P = 0.0000; odds ratio 2.75; 95% confidence interval 1.68–4.52) and 5′(GT)_n allele 2/3 frequency (P = 0.0000; odds ratio 2.69; 95% confidence interval 1.61–4.47) suggesting that SLC11A1 might be a plausible candidate gene for SA.     

Preparation and <Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Microparticle Formulations Containing Meloxicam

In this study, we have formulated chitosan-coated sodium alginate microparticles containing meloxicam (MLX) and aimed to investigate the correlation between in vitro release and in vivo absorbed percentages of meloxicam. The microparticle formulations were prepared by orifice ionic gelation method with two different sodium alginate concentrations, as 1% and 2% (w/v), in order to provide different release rates. Additionally, an oral solution containing 15 mg of meloxicam was administered as the reference solution for evaluation of in vitro/in vivo correlation (ivivc). Following in vitro characterization, plasma levels of MLX and pharmacokinetic parameters [elimination half-life (t _1/2), maximum plasma concentration (C _max), time for C _max (t _max)] after oral administration to New Zealand rabbits were determined. Area under plasma concentration–time curve (AUC_0–∞) was calculated by using trapezoidal method. A linear regression was investigated between released% (in vitro) and absorbed% (in vivo) with a model-independent deconvolution approach. As a result, increase in sodium alginate content lengthened in vitro release time and in vivo t _max value. In addition, for ivivc, linear regression equations with r ² values of 0.8563 and 0.9402 were obtained for microparticles containing 1% and 2% (w/v) sodium alginate, respectively. Lower prediction error for 2% sodium alginate formulations (7.419 ± 4.068) compared to 1% sodium alginate formulations (9.458 ± 5.106) indicated a more precise ivivc for 2% sodium alginate formulation.     

Growth of <Emphasis Type="Italic">Pseudomonas chloritidismutans</Emphasis> AW-1<Superscript>T</Superscript> on <Emphasis Type="Italic">n</Emphasis>-alkanes with chlorate as electron acceptor

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1^T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1^T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day⁻¹, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Cytogenetic characterization of <Emphasis Type="Italic">Hydrangea involucrata</Emphasis> Sieb. and <Emphasis Type="Italic">H. aspera</Emphasis> D. Don complex (Hydrangeaceae)<Emphasis Type="Italic">:</Emphasis> genetic,evolutional, and taxonomic implications

The subsection Asperae of genus Hydrangea L. (Hydrangeaceae) has been investigated for three reasons: several ambiguous classifications concerning Hydrangea aspera have been published, unexpected differences in genome size among seven accessions have been reported Cerbah et al. (Theor Appl Genet 103:45–51, 2001), and two atypical chromosome numbers (2n = 30 for Hydrangea involucrata and 2n = 34 for H. aspera) have been found when all other species of the genus present 2n = 36. Therefore, these two species and four subspecies of Hydrangea in all 29 accessions were analyzed for their genome size, chromosome number, and karyotype features. This investigation includes flow cytometric measurements of nuclear DNA content and bases composition (GC%), fluorochrome banding for detection of GC- and AT-rich DNA regions, and fluorescent in situ hybridisation (FISH) for chromosome mapping of 5 S and 18 S-5.8 S-26 S rDNA genes. In the H. aspera complex, the genome size ranged from 2.98 (subsp. sargentiana) to 4.67 pg/2C (subsp. aspera), an exceptional intraspecific variation of 1.57-fold. The mean base composition was 40.5% GC. Our report establishes the first karyotype for the species H. involucrata, and for the subspecies of H. aspera which indeed present different formulae, offering an element of discrimination. FISH and fluorochrome banding revealed the important differentiation between these two species (H. involucrata and H. aspera) and among four subspecies of the H. aspera complex. Our results are in agreement with the Chinese classification that places the groups Kawakami and Villosa as two different species: Hydrangea villosa Rehder and Hydrangea kawakami Hayata. This knowledge can contribute to effective germplasm management and horticultural use.     

Kinetics of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> growth on high glucose concentrations

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S ₀) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S ₀ values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ _X,m) were not affected with S ₀ up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in μ _X,m values. These higher S ₀ values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X _m = 15.2–15.9 g/l) were achieved with S ₀ over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S ₀ from 30.8 to 152 g/l, cell yield (Y _X/S ) decreased from 0.58 to 0.41 g/g. On the other hand, with S ₀ = 10 g/l growth was limited by substrate, and Y _X/S has shown its maximum value (0.83 g/g).     

Ovarian and Uterine Ultrasonography in <Emphasis Type="Italic">Aotus azarai infulatus</Emphasis>

We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2) was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5–12 MHz linear array probe. The uterine volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females’ mass, RtOV (r = 0.28) and LtOV (r = 0.16).     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.