Stacking of safranine in liposomes during valinomycininduced efflux of potassium ions

Liposomes were prepared from phosphatidylcholine and cardiolipin in a KCl medium and suspended in a choline chloride medium with safranine. When efflux of K⁺ was induced by valinomycin, spectral shifts characteristic of stacking were observed. Ca²⁺ inhibited the rate of stacking in a competitive manner with a K_i of about 200 μM, while La³⁺ was about 10 times more potent. When liposomes were prepared from phospholipids with a higher ratio of cardiolipin to phosphatidyl-choline the inhibition was more potent. No effect on the stacking phenomena was seen when Ca²⁺ was added after the stacking was completed. When Ca²⁺ or an organic cation with four charges, spermine, was trapped in the intraliposomal compartment, no significant change in the rate of stacking was seen. However, the extent of stacking was decreased. It is suggested that safranine is driven by a diffusion potential to a site that is inaccessible to Ca²⁺ in the medium, presumably to the inner boundaries of the liposomal membranes.     

A study on using fireclay as a biomass carrier in an activated sludge system

By adding a biomass carrier to an activated sludge system, the biomass concentration will increase, and subsequently the organic removal efficiency will be enhanced. In this study, the possibility of using excess sludge from ceramic and tile manufacturing plants as a biomass carrier was investigated. The aim of this study was to determine the effect of using fireclay as a biomass carrier on biomass concentration, organic removal and nitrification efficiency in an activated sludge system. Experiments were conducted by using a bench scale activated sludge system operating in batch and continuous modes. Artificial simulated wastewater was made by using recirculated water in a ceramic manufactutring plant. In the continuous mode, hydraulic detention time in the aeration reactor was 8 and 22 h. In the batch mode, aeration time was 8 and 16 h. Fireclay doses were 500, 1,400 and 2,250 mg l⁻¹, and were added to the reactors in each experiment separately. The reactor with added fireclay was called a Hybrid Biological Reactor (HBR). A reactor without added fireclay was used as a control. Efficiency parameters such as COD, MLVSS and nitrate were measured in the control and HBR reactors according to standard methods. The average concentration of biomass in the HBR reactor was greater than in the control reactor. The total biomass concentration in the HBR reactor (2.25 g l⁻¹ fireclay) in the continuous mode was 3,000 mg l⁻¹ and in the batch mode was 2,400 mg l⁻¹. The attached biomass concentration in the HBR reactor (2.25 g l⁻¹ fireclay) in the continuous mode was 1,500 mg l⁻¹ and in the batch mode was 980 mg l⁻¹. Efficiency for COD removal in the HBR and control reactor was 95 and 55%, respectively. In the HBR reactor, nitrification was enhanced, and the concentration of nitrate was increased by 80%. By increasing the fireclay dose, total and attached biomass was increased. By adding fireclay as a biomass carrier, the efficiency of an activated sludge system to treat wastewater from ceramic manufacturing plants was increased.     

Immunoreactivity of thymosin beta 4 in human foetal and adult genitourinary tract

Thymosins beta 4 (Tβ4) is a member of the beta-thymosins family, a family of peptides playing essential roles in many cellular functions. Our recent studies suggested Tβ4 plays a key role in the development of human salivary glands and the gastrointestinal tract. The aim of this study was to analyse the presence of Tβ4 in the human adult and foetal genitourinary tract. Immunolocalization of Tβ4 was studied in autoptic samples of kidney, bladder, uterus, ovary, testicle and prostate obtained from four human foetuses and four adults. Presence of the peptide was observed in cells of different origin: in surface epithelium, in gland epithelial cells and in the interstitial cells. Tβ4 was mainly found in adult and foetal bladder in the transitional epithelial cells; in the adult endometrium, glands and stromal cells were immunoreactive for the peptide; Tβ4 was mainly localized in the glands of foetal prostate while, in the adults a weak Tβ4 reactivity was restricted to the stroma. In adult and foetal kidney, Tβ4 reactivity was restricted to ducts and tubules with completely spared glomeruli; a weak positivity was observed in adult and foetal oocytes; immunoreactivity was mainly localized in the interstitial cells of foetal and adult testis. In this study, we confirm that Tβ4 could play a relevant role during human development, even in the genitourinary tract, and reveal that immunoreactivity for this peptide may change during postnatal and adult life.     

Continuous phenol biodegradation in a simple packed bed bioreactor of calcium alginate-immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> (NCIM 3556)

Phenol biodegradation in a continuous system of immobilized Candida tropicalis NCIM 3556 was studied. The bioreactor was simple, it had a feed inlet from the bottom and the effluent outlet from top, no supplementary oxygen was supplied, the reactor was operated continuously for 116 days. Initially the column was run continuously with a feed concentration of 2 g l⁻¹ for 42 days whence a degradation of >97% was achieved. The feed concentration was then increased to 3 g l⁻¹, for which a ~80% biodegradation was sustained for 90 days after which there was a steady decrease in the performance. When the phenol degradation was reduced to ~50% in 116 days, the reactor was stopped. The efficiency of free cells recycled every 24 h and immobilized cells were compared; it was estimated that repeated reuse of free cells in batch mode gave an overall efficiency of 0.102 g phenol degradation g⁻¹ cell wet weight in 12 days. In contrast, the immobilized system of the same biomass had a longer working lifetime of ~4 months indicating an efficiency of 3.72 g phenol g⁻¹ cell wet wt.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe²⁺ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.     

Molecular Cloning and Characterization of a Mannose-binding Lectin Gene from Dendrobium officinale

Using RNA extracted from Dendrobium officinale young leaves and primers designed according to the conservative regions of Orchidaceae lectins, the full-length cDNA of Dendrobium officinale agglutinin (DOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of doa was 768 bp and contained a 498 bp open reading frame (ORF) encoding a lectin precursor of 165 amino acids. Through comparative analysis of doa gene and its deduced amino acid sequence with those of other Orchidaceae species, it was found that doa encoded a precursor lectin with signal peptide. DOA was a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that doa mRNA expression was detected in all tested tissues including root, stem and leaf, however, the expression was higher in stem, lower in leaf. As the doa mRNA was detected in all the tested plant tissues, the doa was considered to be a constitutively expressed gene.     

Chromosomal relationships of heterochromatin bodies in a moss,Dicranum tauricum Sapehin

Abstract

The largest bivalent in a population of Dicranum tauricum with the haploid chromosome number of n = 12 was found to divide precociously during meiosis. Since it contained no constitutive heterochromatin, as revealed by a Giemsa C-banding technique, it could not be identified as an H-chromosome. A large body of predominantly facultative heterochromatin was recognized in gametophytic mitotic and pre-meiotic interphase cells and was possibly a composite structure. During these stages, a small block of constitutive heterochromatin was associated with the nucleolus. It was related to a chromosome which, because it was not the smallest member of the complement, could not be called an H-chromosome. Consequently, a reassessment of H- and H-chromosomes in mosses is recommended.     

Taxol transport in Taxus baccata cell suspension cultures

Taxol transport in Taxus baccata L. cell suspension cultures was studied using 〚¹⁴C〛-taxol as a tracer. The time course of uptake showed a saturable absorption that reached a maximum within 20 min. The uptake depended on its exogenous concentration and its accumulation was highly stimulated in the presence of 10–15 μM exogenous taxol. The absorbed molecule was found to localise both in the cell walls (20 %) and in the cell protoplasts (80 %), suggesting an accumulation within the vacuoles. Taxol uptake was strongly inhibited by Na-orthovanadate and verapamil, while Ca²⁺ was found to be one of the factors required for the active absorption of the molecule, since in the absence of this cation, the uptake was reduced by about 40 % and occurred mainly through a non-energy dependent mechanism. Taxol release into the culture medium was demonstrated not to depend on cell lysis, occurred through a mechanism that reached its maximum after 10–15 min and was strongly enhanced by treatment with Na-orthovanadate and verapamil, although the effect was found to be transient.     

Patchouli virus X, a new potexvirus from Pogostemon clabin

This work describes a potexvirus obtained from patchouli, Pogostemon clabin, collected in São Paulo, Brazil in 1992. The plants showed mosaic and were infected by a potyvirus and a potexvirus. The potexvirus had a host range limited to Amaranthaceae, Solanaceae and Labiatae and was named Patchouli virus X (PatVX). PatVX was not transmitted by scissors pruning, in tobacco seeds or by Myzus nicotinae. The virus was purified and a specific antiserum with a titre great than 1:512 000 in dot‐ELISA was produced. The virus was serologically related to Papaya mosaic virus, Potato virus X, Viola mottle virus, White clover mosaic virus and Lily virus X. It had a coat protein of 21 071 ± 1 010 Mr. as determined by SDS‐PAGE. Immunolabelling tests demonstrated that fibrillar masses in the cytoplasm contain the coat protein. The presence of a dsRNA was detected in PatVX infected plants.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Studies on the nutrition of the genus Pestalotiopsis II. Chromatographic analysis of the medium during the utilization of various carbohydrates

The utilization of 17 different carbohydrates by four isolates ofPestalotiopsis was studied by the paper chromatographic technique.Among the pentoses, xylose, which was recorded as a poor source, was not consumed even in 15 days by any of the isolates. L-arabinose was exhausted in 6–8 days while L-rhamnose was used up in 9–10 days, both supporting growth equal to glucose as a source. There was a good correlation between the efficiency and the rate of utilization of the three pentose sugars.The utilization of the hexoses had no correlation with dry weight output as was in the case of the pentoses. Glucose from the medium was exhausted between 4–9 days; fructose required 7–10 days. Fructose was much superior than glucose but was consumed slowly as compared to glucose. D-galactose which was a poor source was used up only in 7 days byP. royneae but the other three isolates took longer time, 11–14 days to exhaust the sugar. L-sorbose was used up in 10–15 days. D-mannose, which was a good source for mycelial attainment required 6–12 days for complete utilization.Hydrolytic pathway of utilization of oligosaccharides was recorded for all the isolates; in lactose, however, the component sugars could not be detected and the disaccharide was present up to the end of the incubation period. This was obviously due to slow rate of its breakdown. Sucrose was hydrolysed in the shortest period of 2 days in all cases. Glucose was utilized faster than the other hydrolytic products by three isolates.P. royneae, however, finished both in equal time of 6 days. Maltose, cellobiose and trehalose were used slowly. In maltose, one or two transient oligosaccharides were also detected. Trehalose persisted for the longest time. Melibiose was present till the end of the incubation period during the growth ofP. gracilis andP. paeoniae isolate I. The isolate II ofP. paeoniae andP. royneae consumed it in 9 days. Only one of its hydrolytic products, i.e. glucose was detected in the medium, on raffinose solution only fructose and galactose were spotted.The hydrolytic products of starch could not be detected in the medium. In dextrin, however, glucose was detected in the medium used by three isolates other thanP. paeoniae isolate I.Oligosaccharide synthesis was accomplished by all the isolates ofPestalotiopsis under study. On maltose medium,P. gracilis andP. paeoniae isolate I synthesised two such transient oligosaccharides,P. royneae andP. paeoniae II formed only one.     

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Putative down-stream signaling molecule of GTPase in Porphyromonas gingivalis

Porphyromonas gingivalis is a strict anaerobic bacterium mainly responsible for periodontal disease in oral cavity. Putative GTPase gene (pgp) of this bacterium was cloned and its recombinant protein (rPGP) was produced in Escherichia coli. Based on the amino acid sequence of SGP that is a GTP-binding protein of Streptococcus mutans, putative GTPase amino acid sequence was deduced in the data base of genome sequences of Porphyromonas gingivalis. A 900-bp PCR fragment was amplified with P. gingivalis genomic DNA as a template and cloned into E. coli JM109. Then pgp was transferred into pQE-30 expression vector to make pQE-PGP for production of rPGP. This protein was produced and purified by Ni-NTA affinity column chromatography. Anti-PGP antibody was also produced in Sprague Dawley rats. Using Westernblot analysis with this antibody, it was confirmed that the rPGP produced in E. coli was identical to that of donor strain. Furthermore, by Southernblot analysis it was revealed that the pgp was originated from P. gingivalis. By immunoprecipitation with anti-PGP antibody and N-terminal amino acid sequence analysis it was found that PGP was able to bind to acetate kinase, which was reported to be a secondary signaling molecule in anaerobic microorganisms. Therefore, these results imply that P. gingivalis produces putative GTPase and this protein might play a potential role in signaling pathway in oral biofilm formation.     

Hydrostatic and osmotic pressure activated channel in plant vacuole

The vacuolar membrane of red beet vacuoles contains a channel which was not gated by voltage or Ca²⁺ ions. Its unit conductance was 20 pS in 200 mM symmetrical KCl solutions. It was stretch activated: the conductance remained constant but the probability of opening was increased by suction or pressure applied to a membrane patch. A 1.5-kNm^-2 suction applied to isolated patches or a 0.08-kNm^-2 pressure applied to a 45-μm diameter vacuole induced an e-fold change in the mean current. A 75% inhibition of the channel current was obtained with 10 μM Gd³⁺ on the cytoplasmic side. The channel was more permeable for K⁺ than for Cl^- (P_K/P_Cl ~ 3). A possible clustering for this channel was suggested by the recordings of the patch current. The channel properties were not significantly affected by a change in sorbitol osmolality in the solutions under isoosmotic conditions, between 0.6 and 1 mol/kg sorbitol. However, the channel was very sensitive to an osmotic gradient. A 0.2-mol/kg sorbitol gradient induced a two-fold increase in unit conductance and a thirty-fold increase in the mean patch current of the channel. A current was measured, when the osmotic gradient was the only driving force applied to the vacuolar membrane. The hydrostatic and osmotic pressure (HOP) activated channel described in this paper could be gated in vivo condition by a change in osmolality, without the need of a change in the turgor pressure in the cell. The HOP channel represents a possible example of an osmoreceptor for plant cells.     

Influence of fungi on growth and survival of Onychiurus armatus (Collembola) in a metal polluted soil

Summary The influence of food quantity and quality on growth and survival of Onychiurus armatus (Tullb.) in metal polluted environments has been investigated in laboratory experiments. The Collembola was reared on five species of fungi isolated from a metal polluted soil close to a brass mill in SE Sweden.Survival of O. armatus was improved when fungal biomass was continuously added in a polluted mor (1,300 ppm Zn and 200 ppm Cu), and when specimens were fed metal polluted fungi for 1, 3 and 7 days a week, only those that were starved had increased mortality. Allometric growth, on the other hand, was significantly reduced when Collembola was given surplus of metal polluted fungi, whereas growth losses caused by metals were offset by protein rich food. Hence, sufficient food quantities alone could overcome mortality losses but not growth retardation in a metal polluted environment.Feeding preference of O. armatus was not determined by the protein content of the fungi although this was beneficial for growth. Metals changed the relative palatability of fungal species, but one of the metal tolerant species, Paecilomyces farinosus, which was also protein rich, remained reasonably attractive for O. armatus also when it was metal polluted. The mechanisms by which growth and survival of O. armatus were promoted by a combination of protein and Zn/Cu rich fungi seemed to be crucial in understanding the fate of a population of this species in a metal polluted soil.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Distance effect of co-occurring tree species on pine wilt disease incidence inPinus densiflora seedlings inoculated withBursaphelenchus xylophilus

Field-grownPinus densiflora seedlings were inoculated withBursaphelenchus xylophilus and the incidence of pine wilt disease was analyzed with respect to the spatial relationships between pine seedlings and adjacent seedlings of other tree species in a pure pine stand and three stands mixed withAlnus sieboldiana, Sarothamus scoparius or naturally associated species. The disease incidence was 60.9% in a 0–40 cm distance class from the nearestA. sieboldiana seedlings and then decreased with increasing minimum distance. The mean minimum distance between pine seedlings andA. sieboldiana was significantly shorter in diseased seedlings than in healthy ones. The highest disease incidence (53.9%) was found in a 0–40 cm distance class from the nearestS. scoparius, although the relationship with minimum distance was blurred by a high incidence in the 80-cm distance class. The mean minimum distance between pine seedlings was significantly short in diseased seedlings in a pure stand, whereas it was not so short between pine seedlings and other tree species in the two stands mixed withS. scoparius and the naturally associated species. The distance effect on disease incidence was noticeable inA. sieboldiana andP. densiflora at high density. This was not so clear inS. scoparius and was not found in the naturally associated species.     

The possible mode of action of prostaglandins: X. Antagonism between prostaglandin F2a and prolactin or human chorionic gonadotropin : A comparative study

A comparative assessment of the efficacy of prolactin or HCG in reversing the luteolytic property of a single dose (2.0 mg/kg) of PGF₂a was conducted in pregnant (day 10) rats. It was found that prolactin maintained pregnancy in 90% of the test animals. HCG though found to maintain pregnancy in 70% of the PGF₂a-treated pregnants, the fetal survival rate was, however, recorded to be 62% against the control value of 98%. Conversely, prolactin replacement maintained the fetal survival rate as high as 93.4%. Moreover, the growth of the fetuses, placentae and corpus luteum in the pregnants having prolactin in conjunction with PGF₂a was also found to be greater compared to the animals which h ad a combined regimen of PGF₂a and HCG, but identical to controls. On the other hand, similar combined regimen when applied to hysterectomized pregnant rats, it was observed that though the vaginal diestrus was maintained by the prolactin or HCG in the presence of PGF₂a, the prolactin regimen was found to be superb compared to HCG in the maintenance of luteal weight and functional activity. It was concluded that the antifertility effect of PGF₂a in the rat is primarily the consequence of luteolysis and prolactin seems to be a much more appropriate hormonal replacement compared to HCG, a long-acting LH, in antagonizing the luteolytic property of PGF₂a.