Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Quantitative aspects of the interaction between ouabain and (Na + K)-activated ATPase in vitro

The inhibitory effect of ouabain on (Na⁺ + K⁺)-activated ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na⁺, Mg²⁺ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na⁺ + K⁺)-activated ATPase reaction was subsequently started by the addition of a KCl solution.     

Nutrient composition and in vitro ruminal fermentation of tropical legume mixtures with contrasting tannin contents

Various combinations of a low-tannin herbaceous legume (Vigna unguiculata) and foliage of tanniniferous shrub legumes (Calliandra calothyrsus, Flemingia macrophylla and Leucaena leucocephala) or a low-tannin shrub legume (Cratylia argentea), all mixed together with a low-quality tropical grass (Brachiaria humidicola), were tested in vitro for differences in the effects on ruminal fermentation. Two experiments with the gas transducer technique were carried out, where each forage mixture was tested either with or without polyethylene glycol in order to be able to identify tannin-related effects (n = 3). In Experiment 1, a stepwise replacement of V. unguiculata by C. calothyrsus (5:0, 4:1, 3:2, 2:3, 1:4, 0:5) at a legume proportion of 1/3 or 2/3 in the mixture was evaluated. Together with two grass-alone and four pure legume treatments this added up to 30 treatments. In Experiment 2, V. unguiculata was gradually replaced by each of the four shrub legumes (3:0, 2:1, 1:2, 0:3) in grass–legume ratios of 2:1, adding up, together with two grass-alone treatments, to 28 treatments. When added alone, V. unguiculata resulted in high fermentative activity as measured by gas production and kinetics as well as low proportion of undegraded crude protein. When V. unguiculata was replaced by the low-tannin C. argentea in Experiment 2, there was no noticeable difference (P>0.05) in fermentative activity. In both experiments, the effect of the substitution of V. unguiculata by tanniniferous shrub legumes resulted in a declining gas production and an increasing proportion of undegraded crude protein (P<0.001). However, the extent of these changes depended on the level of replacement and the shrub legume species (P<0.001). The results of Experiment 2 illustrate that this was the consequence not only of different tannin contents (less adverse effects with L. leucocephala than with C. calothyrsus) but also differences in the chemical properties of the tannins present in these shrub legume species (much less adverse effects with L. leucocephala than with F. macrophylla despite similar tannin contents). Furthermore these results indicate that, once the extent of the effects of a tanniniferous legume is known, one may calculate the maximal level of replacement of a low-tannin legume in a grass diet possible without negative effects on ruminal fermentation. This allows to improve dry season grass-based diets with as few as possible of the expensive and less well growing low-tannin legume.     

Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V₁ portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA₃-B₃ heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA₃-B₃ forming V₁ (NtpA₃-B₃-D-G) complex independent of nucleotides. The V₁ formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V₁ complex was as high as that of native V₁-ATPase purified from the V₀V₁ complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V₁ complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V₁-ATPase complex.     

Synthesis, characterization and in vitro cytotoxicity studies of platinum(IV) complexes with thiouracil ligands

Reactions of [PtMe₃(bpy)(Me₂CO)][BF₄] (2) with the thionucleobases 2-thiouracil (s²Ura), 4-thiouracil (s⁴Ura) and 2,4-dithiouracil (s²s⁴Ura) resulted in the formation of complexes of the type [PtMe₃(bpy)(L-κS)][BF₄] (L = s²Ura, 3; s⁴Ura, 4; s²s⁴Ura, 5). The complexes were characterized by NMR spectroscopy (¹H, ¹³C, ¹⁹⁵Pt), IR spectroscopy as well as microanalyses. The coordination through the C4S groups (4, 5) was additionally confirmed by DFT calculations, where it was shown that these complexes [PtMe₃(bpy)(L-κS⁴)]⁺ (L = s⁴Ura, s²s⁴Ura) are about 5.8 (4b) and 3.3 kcal/mol (5b), respectively, more stable than the respective complexes, having thiouracil ligands bound through the C2X groups (X = O, 4a; S, 5a). For [PtMe₃(bpy)(s²Ura-κS²)][BF₄] (3) no preferred coordination mode could be assigned solely based on DFT calculations. Analysis of NMR spectra showed the κS² coordination. In vitro cytotoxic studies of complexes 3−5 on nine different cell lines (8505C, A253, FaDu, A431, A549, A2780, DLD-1, HCT-8, HT-29) revealed in most cases moderate activities. However, 3 and 5 showed significant activity towards A549 and A2780, respectively, possessing IC₅₀ values comparable to those of cisplatin. Cell cycle perturbations and trypan blue exclusion test on cancer cell line A431 using [PtMe₃(bpy)(s²s⁴Ura-κS⁴)][BF₄] (5) showed induction of apoptotic cell death. Furthermore, the reaction of [PtMe₃(OAc-κ²O,O′)(Me₂CO)] (6) with 4-thiouracil yielded the dinuclear complex [(PtMe₃)₂(μ-s⁴Ura_-H)₂] (7), which has been characterized by microanalysis, NMR (¹H, ¹³C, ¹⁹⁵Pt) and IR spectroscopy as well as ESI mass spectrometry. X-ray diffraction analysis of crystals yielded in an isolated case exhibited the presence of a hexanuclear thiouracilato platinum(IV) complex, possessing each three different kinds of methyl platinum(IV) moieties and 4-thiouracilato ligands. This exhibited the ability of 4-thiouracil platinum(IV) complexes to form multinuclear complexes.     

Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803

Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

In sacco degradation kinetics of fresh and field-cured peanut (Arachis hypogaea L.) forage harvested at different maturities

There is interest in growing peanut (Arachis hypogaea L.) for forage, but little is known about the nutritive value and forage quality of modern cultivars. The objective of this study was to compare the chemical composition and in sacco degradation kinetics of three cultivars of peanuts (cv. ‘C99-R’, ‘Georgia-01R’, and ‘York’) at either stage 2 or 8 maturities when fresh and field-cured. Herbage yield was at least 3000 kg DM/ha for all cultivars at both maturities. Crude protein (CP) was greater (P < 0.0001) at R2 stage than at R8 stage; whereas, neutral detergent fiber (aNDF), acid detergent fiber, and Lignin (sa) were greater (P < 0.01) at R8 than R2 maturity stages. Water soluble carbohydrate and acid detergent insoluble nitrogen was not different (P > 0.07) among cultivars, maturity stage, or harvest forms. In vitro true digestibility was greatest (P < 0.02) for C99-R and least for York. Undegradable intake protein concentration was greatest (P < 0.04) in York and least for C99-R. Maturity had a greater effect on the degradation kinetics than harvest form or cultivar. The dry matter (DM) and CP in the soluble wash fraction (A) and insoluble but degradable fraction (B) and the effective ruminal degradability were greater among all cultivars and both harvest forms of the R2 maturity stage than the R8. The undegradable DM, aNDF, and CP in the undegradable fraction were greatest (P < 0.002) for all three cultivars at R8 maturity. The rate of degradation of DM and CP in the B fraction was faster (P < 0.001) at R2 stage than at R8 stage; whereas, rate of aNDF degradation was not different (P > 0.09) among treatments. Lag of DM, aNDF, or CP degradation was not different (P > 0.1) among treatments. The cultivars C99-R and Georgia-01R are recommended for further feeding trials.     

Pollen morphology of the genus Salvia L. (Lamiaceae) in Turkey

Pollen grains of 30 taxa of the genus Salvia, belonging to sections Salvia, Horminum, Drymosphace, Plethiosphace and Hemisphace from Turkey were examined by light (LM) and scanning electron microscopy (SEM). Detailed pollen morphological characteristics are provided for these taxa. Among the studied taxa, S. verticillata subsp. verticillata from sect. Hemisphace has the smallest pollen grains, and S. pachystachys from sect. Salvia possesses the largest ones. The basic shape of the pollen grains in most taxa is suboblate, oblate-spheroidal or prolate-spheroidal. However subprolate pollen grains are recorded for S. macrochlamys from sect. Salvia. The grains are hexacolpate in all taxa, but in S. recognita from sect. Salvia also octacolpate pollen was found. Three distinct exine sculpturing types exist, reticulate-perforate (the common type), reticulate-granulate and bireticulate. The reticulate-perforate and bireticulate sculpturing patterns can be divided into subtypes based on the number of perforations and the number of secondary lumina in each primary lumen. Pollen morphological characteristics of the taxa studied are compared and discussed on the basis of taxonomical concepts. In some cases, these characters are useful in distinguishing the sections. For instance, the presence of 1-2 large central secondary lumina per primary lumen is a significant character of sect. Horminum separating it from the other sections. As well, the presence of holes on colpus membrane ornamentation can be used as a diagnostic taxonomic character for sectional division between sect. Hemisphace and others. S. ballsiana from sect. Salvia is clearly distinct from the other taxa examined by its unique pollen morphology. Further, for several macromorphologically similar taxa pollen structures provide additional evidence to delimite them from each other.     

Influence of tris(2,3-dibromopropyl) isocyanurate on the expression of photosynthesis genes of Nannochloropsis sp.

The proteins encoded by psaA and psaB form a heterodimer, an essential compound of photosystem; while the protein encoded by psbC binds with chlorophyll a in photosystem II, serving as antennae in photosystem. Here we report that a heterocyclic brominated flame retardant, tris(2,3-dibromopropyl) isocyanurate (TBC), inhibited the expression of psaA and psbC, then leads to the decrease of Nannochloropsis sp.'s growth biomass. TBC exposure inhibited the expression of psaA and psbC at 10, 100 ng/mL slightly and 1000 ng/mL significantly. In addition, TBC was found to slow down the growth of Nannochloropsis sp. at concentrations ranging from 10 to 1000 ng/mL. These results indicated that TBC influenced both photosynthesis and growth performance of Nannochloropsis sp.     

Morphology and anatomy of blackberry pyrenes (Rubus L., Rosaceae) Elementary studies of the European representatives of the genus Rubus L.

This article presents the results of studies on the pyrenes of selected European brambles belonging to different subgenera and sections of the genus Rubus. Differences between the pyrenes of particular species are mainly visible in their shape, size and the outer endocarp sculpture. Numerous SEM photographs revealed that the differentiation of the endocarp surface is due to variations in a thin layer of transition sclereids developing between the outer endocarp and a mesocarp parenchyma. The endocarp structure of the genus Rubus can be used in determining some species belonging to different subgenera, even if generally its significance in the taxonomy of the European Rubus is limited.     

Evolutionary aspects of a unique internal mitochondrial targeting signal in nuclear-migrated rps19 of sugar beet (Beta vulgaris L.)

The endosymbiotic theory postulates that many genes migrated from endosymbionts to the nuclear genomes of their hosts. Some migrated genes lack presequences directing proteins to mitochondria, and their mitochondrial targeting signals appear to be inscribed in the core coding regions as internal targeting signals (ITSs). ITSs may have evolved after sequence transfer to nuclei or ITSs may have pre-existed before sequence transfer. Here, we report the molecular cloning of a sugar beet gene for ribosomal protein S19 (Rps19; the first letter is capitalized when the gene is a nuclear gene). We show that sugar beet Rps19 (BvRps19) is an ITS-type gene. Based on amino-acid sequence comparison, dicotyledonous rps19s (the first letter is lower-cased when the gene is a mitochondrial gene), such as tobacco rps19 (Ntrps19), resemble an ancestral form of BvRps19. We investigated whether differences in amino-acid sequences between BvRps19 and Ntrps19 were involved in ITS evolution. Analyses of the intracellular localization of chimaeric GFP-fusion proteins that were transiently expressed in Welsh onion cells showed that Ntrps19-gfp was not localized in mitochondria. When several BvRps19-type amino acid substitutions, none of which was seen in any other angiosperm rps19, were introduced into Ntrps19-gfp, the modified Ntrps19-gfp became localized in mitochondria, supporting the notion that an ITS in BvRps19 evolved following sequence transfer to nuclei. Not all of these substitutions were seen in other ITS-type Rps19s, suggesting that the ITSs of Rps19 are diverse.     

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA^His differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.     

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

Systemic acquired resistance in Cavendish banana induced by infection with an incompatible strain of Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fact that there are no economically viable biological, chemical, or cultural measures of controlling the disease in an infected field leads to search for alternative strategies involving activation of the plant's innate defense system. The mechanisms underlying systemic acquired resistance (SAR) are much less understood in monocots than in dicots. Since systemic protection of plants by attenuated or avirulent pathogens is a typical SAR response, the establishment of a biologically induced SAR model in banana is helpful to investigate the mechanism of SAR to Fusarium wilt. This paper described one such model using incompatible Foc race 1 to induce resistance against Foc tropical race 4 in an in vitro pathosystem. Consistent with the observation that the SAR provided the highest level of protection when the time interval between primary infection and challenge inoculation was 10 d, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL, EC 4.3.1.5), peroxidase (POD, EC 1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), and superoxide dismutase (SOD, EC 1.15.1.1) in systemic tissues also reached the maximum level and were 2.00–2.43 times higher than that of the corresponding controls on the tenth day. The total salicylic acid (SA) content in roots of banana plantlets increased from about 1 to more than 5 μg g⁻¹ FW after the second leaf being inoculated with Foc race 1. The systemic up-regulation of MaNPR1A and MaNPR1B was followed by the second up-regulation of PR-1 and PR-3. Although SA and jasmonic acid (JA)/ethylene (ET) signaling are mostly antagonistic, systemic expression of PR genes regulated by different signaling pathways were simultaneously up-regulated after primary infection, indicating that both pathways are involved in the activation of the SAR.