Studies on the effect of iron overload on rat cortex synaptosomal membranes

Iron as ferrous ammonium sulfate was injected into the cerebral spinal fluid of rats. After three consecutive days of injection of 4 mumol of iron, the total iron content of brain cortex synaptosomes from the iron-treated animals was 2-fold higher than that from control animals receiving the saline vehicle only. Spin label studies of the synaptosomal membranes demonstrated that the lipid region of the membranes became more rigid and, in addition, the mobility of labeled SH groups of membrane proteins decreased after the iron treatment. The cholesterol content was significantly higher in iron-treated animals as compared to controls. Centrophenoxine pretreatment (100 mg/kg body weight daily for 6 weeks) diminished the iron effects. Synaptosomal membrane alterations observed after iron treatment were similar to changes observed previously during aging. This lends support to the notion that free-radical induced damage occurs in brain membranes with increasing age.     

The influence of water level on macrophyte growth and trophic interactions in eutrophic Mediterranean shallow lakes: a mesocosm experiment with and without fish

1. Water‐level fluctuations are typical of lakes located in the semi‐arid Mediterranean region, which is characterised by warm rainy winters and hot dry summers. Ongoing climate change may exacerbate fluctuations and lead to more severe episodes of drought, so information on the effects of water level on the functioning of lake ecosystems in such regions is crucial. 2. In eutrophic Lake Eymir, Turkey, we conducted a 4‐month (summer) field experiment using cylindrical 0.8‐m‐ (low‐water‐level) and 1.6‐m‐deep (high‐water‐level) mesocosms (kept open to the sediment and atmosphere). Fish (tench, Tinca tinca, and bleak, Alburnus escherichii) were added to half of the mesocosms, while the rest were kept fishless. Ten shoots of Potamogeton pectinatus were transplanted to each mesocosm. 3. Sampling for physicochemical variables, chlorophyll a (chl‐a), zooplankton and per cent plant volume inhabited (PVI%) by macrophytes was conducted weekly during the first 5 weeks, and subsequently biweekly. Macrophytes were harvested on the last sampling date. During the course of the experiment, the water level decreased by 0.41 ± 0.06 m. 4. Throughout the experiment, fish affected zooplankton abundance (?), nutrient concentrations (+), chl‐a (+) and water clarity (?) most strongly in the low‐water‐level mesocosms and the zooplankton community shifted towards dominance of small‐sized forms. The fishless mesocosms had a higher zooplankton/phytoplankton ratio, suggesting higher grazing. 5. Greatest macrophyte growth was observed in the low‐water‐level fishless mesocosms. However, despite high nutrient concentrations and low water clarity, macrophytes were also abundant in the fish mesocosms and particularly increased following a water‐level decrease from midsummer onwards. Macrophyte growth was poor in the high‐water‐level mesocosms, even in the fishless ones with high water clarity. This was ascribed to extensive periphyton development reducing light availability for the macrophytes. 6. Our results indicate that a reduction in water level during summer may help maintain the growth of macrophytes in Mediterranean eutrophic shallow lakes, despite a strong negative effect of fish predation on water clarity. It is therefore probable that an expected negative effect of global climate change on water clarity because of eutrophication and enhanced top‐down control of fish may be, at least partly, counteracted by reduced water level, provided that physical disturbance is not severe.     

Solution equilibria and structural characterisation of the palladium(II) and mixed metal complexes of peptides containing methionyl residues

Palladium(II) complexes of the peptides GlyMet, GlyMetGly and GlyGlyMet containing methionyl residues were studied by potentiometric and 1H NMR spectroscopic methods. The coordination of terminal amino and deprotonated amide nitrogen and thioether sulfur donor atoms was suggested in the mono complexes of GlyMet and GlyMetGly. The fourth coordination site of these complexes can be occupied by solvent molecule, chloride or hydroxide ions or by another ligand molecule in the bis or mixed ligand complexes. The second ligand coordinates monodentately via the thioether function in acidic media and the amino group under neutral or basic conditions. The stoichiometry of the major species formed in the palladium(II)-GlyGlyMet system is [PdH(-2) L]- and this is coordinated by the amino, two-amide and the thioether donor functions. Thioether bridged mixed metal complexes formed in the reaction of [Pd(dien)]2+ and [Cu(GlyMetH(-1))] or [Ni(GlyMetGlyH(-2))]- also have been detected by spectroscopic techniques.     

Highly Sensitive Spectrofluorimetric Method for Determination of Certain Aminoglycosides in Pharmaceutical Formulations and Human Plasma

A simple, reliable, highly sensitive and selective spectrofluorimetric method has been developed for determination of certain aminoglycosides namely amikacin sulfate, tobramycin, neomycin sulfate, gentamicin sulfate, kanamycin sulfate and streptomycin sulfate. The method is based on the formation of a charge transfer complexes between these drugs and safranin in buffer solution of pH 8. The formed complexes were quantitatively extracted with chloroform under the optimized experimental conditions. These complexes showed an excitation maxima at 519–524 nm and emission maxima at 545–570 nm. The calibration plots were constructed over the range of 4–60 pg mL⁻¹ for amikacin, 4–50 pg mL⁻¹ for gentamicin, neomycin and kanamycin, 4–40 pg mL⁻¹ for streptomycin and 5–50 pg mL⁻¹ for tobramycin. The proposed method was successfully applied to the analysis of the cited drugs in dosage forms. The proposed method was validated according to ICH and USP guidelines with respect to specificity, linearity, accuracy, precision and robustness. The high sensitivity of the proposed method allowed determination of amikacin and gentamicin in spiked and real human plasma.Key words: aminoglycosides, dosage forms, human plasma, safranin, spectrofluorimetry     

Experimental Infection of New Zealand White Rabbits (Oryctolagus cuniculi) with Leporid herpesvirus 4

Leporid herpesvirus 4 (LHV4) is a novel alphaherpesvirus recently identified in domestic rabbits (Oryctolagus cuniculi). Little is known about the pathogenesis or time course of disease induced by this virus. We therefore intranasally inoculated 22 female New Zealand white rabbits with 8.4 × 10⁴ CCID₅₀ of a clinical viral isolate. Rabbits were monitored for clinical signs, viral shedding in oculonasal secretions, and development and persistence of serum antibodies. Rabbits were euthanized at 3, 5, 7, 14, and 22 d postinfection (dpi) to evaluate gross and microscopic changes. Clinical signs were apparent between 3 to 8 dpi, and included oculonasal discharge, respiratory distress, and reduced appetite, and viral shedding occurred between 2 and 8 dpi. Seroconversion was seen at 11 dpi and persisted to the end of the study (day 22). Severe necrohemorrhagic bronchopneumonia and marked pulmonary edema were noted by 5 dpi and were most severe at 7 dpi. Pulmonary changes largely resolved by 22 dpi. In addition, multifocal splenic necrosis was present at 5 dpi and progressed to submassive necrosis by 7 dpi. Eosinophilic herpesviral intranuclear inclusion bodies were detected in the nasal mucosa, skin, spleen, and lung between 3 to 14 dpi. LHV4 is a pathogen that should be considered for rabbits that present with acute respiratory disease. LHV4 infection can be diagnosed based on characteristic microscopic changes in the lungs and spleen and by virus isolation. Serum antibody levels may be used to monitor viral prevalence in colonies.Abbreviations: CCID₅₀, 50% cell culture infectious dose; CRFK, Crandall feline kidney; dpi, days post infection; LHV4, Leporid herpesvirus 4; RHDV, rabbit hemorrhagic disease virus; S:P, sample:positiveHerpesviridae is a large family of enveloped, double-stranded DNA viruses within the order Herpesvirales. Viruses within this order are morphologically similar, possessing large genomes ranging from 125 to 290 kb. The linear DNA is packaged within an icosahedral capsid that is surrounded by a proteinaceous tegument and enclosed in a lipid envelope.¹⁰ The family Herpesviridae comprises more than 100 different virus species that infect mammals, birds, and reptiles. The family is divided into 3 subfamilies—alphaherpesviruses, betaherpesviruses, and gammaherpesviruses—according to distinguishing biologic properties of the viruses. Alphaherpesviruses demonstrate rapid lytic responses in cell culture, whereas betaherpesviruses are slow-growing, often producing giant cells in tissue, and gammaherpesviruses typically infect lymphoid tissue, leading to oncogenesis. Division into subfamilies on the basis of biologic behavior is also consistent with phylogenetic analysis.²⁹ Phylogenetic trees of the viral species are used to further subclassify viruses into genera, which typically closely follow the evolution of the host species.²³There are 4 known herpesviruses of rabbits. Leporid herpesvirus 1 (cottontail herpesvirus) and Leporid herpesvirus 3 (Herpesvirus sylvilagus) are gammaherpesviruses that have been isolated from wild cottontail rabbits (Sylvilagus floridanus). Both LHV1 and LHV3 were isolated incidentally from primary kidney cell cultures during searches for papillomaviruses⁸ and other viruses.¹³ The minor differences in immunoreactivity between LHV1 and LHV3 suggest that they are unique viruses, but complete genetic analyses are unavailable.⁶ LHV3 infection can induce lymphoproliferative disease and neoplasia in cottontail rabbits,¹² but the virus is unable to establish productive infection in domestic rabbits, such as New Zealand white rabbits (Oryctolagus cuniculi).¹⁴ No infection trials with LHV1 have been reported. Leporid herpesvirus 2, also known as virus 3 and Herpesvirus cuniculi, is a gammaherpesvirus that was first isolated in domestic laboratory rabbits during the search for the causative agent of chickenpox.³¹ LHV2 was later isolated as a slow-growing contaminant of a primary rabbit kidney cell culture.²⁵ Early infection studies with LHV2 demonstrated induction of neurologic signs, including nonsuppurative encephalitis with classic herpetic intranuclear inclusion bodies, after intracerebral inoculation.³¹ Recent studies suggest the histologic evidence of a mild, subclinical encephalitis after infection of New Zealand white rabbits.³⁷ Natural infections of Human herpesvirus 1 (herpes simplex 1) have been reported in rabbits, resulting in fatal encephalitis.^24,32,36Leporid herpesvirus 4 (LHV4) is a novel herpesvirus that was independently diagnosed and isolated from commercial rabbits in Alaska¹⁹ and a pet rabbit in northern Ontario.⁵ In 1990, cases of rabbit disease with similar clinical signs were reported among commercial meat rabbits in Alberta and British Columbia; the etiologic agent in these 2 cases was identified as a herpesvirus, but further genetic analyses were not performed.^27,33 Affected rabbits show variable clinical signs including lethargy, anorexia, conjunctivitis, fever, and abortion. Predominant pathologic findings include hemorrhagic dermatitis, splenic necrosis, hepatic necrosis, and multifocal pulmonary hemorrhage and edema. Distinctive glassy eosinophilic herpetic intranuclear inclusion bodies were observed in the skin and mesenchymal cells of the spleen and lung.^5,18 Postinfection morbidity and mortality have been reported to be 50% and 20%, respectively. However, the clinical disease and time course have not been studied previously, and only anecdotal reports from veterinary clients have been described. On the basis of its rapid growth and cytopathic effect in cell culture, LHV4 is classified as an alphaherpesvirus. Phylogenetic analysis of multiple genes has indicated that LHV4 segregates to the genus Simplexvirus,^2,18 which is unusual because this genus consists primarily of primate herpesviruses. The only other nonprimate species in this family are Bovine herpesvirus 2, Macropodid herpesvirus 1, and Macropodid herpesvirus 2,¹⁰ suggesting that these viral species may have migrated from primates, such as human caregivers, to these other species.²²With the emergence of a newly recognized infectious disease of rabbits, it is important to consider its effect on all domestic rabbit populations, including those used in research. In addition to welfare concerns, infectious disease can introduce unacceptable variability in research data.²¹ Guidelines for the care and use of laboratory animals from the Canadian Council on Animal Care and National Academy of Sciences indicate a need for the surveillance and eradication of known pathogens.^7,15 Furthermore, many studies use rabbits to answer research questions about other alphaherpesviruses. The rabbit is a popular model for the ocular keratitis induced by Herpes simplex virus 1 ⁹ and for the study of Bovid herpesvirus 1 and Bovid herpesvirus 5.³⁴ In addition, rabbits have been used in investigations of Macacine herpesvirus 1 (B virus) infections.⁴ As illustrated with the discovery of LHV2, rabbit cell cultures are often used to isolate viruses.^25,31 The presence of either active or latent LHV4 infection could seriously affect the interpretation of findings from these studies. It is imperative that laboratory animal veterinarians be aware of LHV4 and be able to identify and diagnose infections in rabbits.A preliminary investigation of a suspected herpesvirus infection in 2 domestic New Zealand white rabbits demonstrated splenic and hepatic necrosis, pulmonary congestion and edema, and necrosis at the site of inoculation.²⁷ After the initial discovery of LHV4, a few young New Zealand white rabbits were experimentally inoculated both intranasally and intracorneally with very large doses of virus and developed conjunctivitis and systemic illness.¹⁸ Pathology was conducted only on one animal at the peak of infection, and revealed splenic and lymph node necrosis.¹⁸ Whether rabbits can recover from the disease, how long they shed virus after infection, and if and when protective antibody titers develop were not evaluated.In the current study, we characterized the progression of clinical signs and the gross and microscopic changes after the intranasal inoculation of adult female New Zealand white rabbits with a sublethal dose of LHV4. We further evaluated viral shedding, neutralizing antibody production, and the recovery of rabbits from infection. Although bacterial infection may contribute to the progression and severity of disease in pet or commercial rabbit settings, we used SPF rabbits to isolate the direct pathologic effects of LHV4. This study provides essential information to veterinarians for the diagnosis of LHV4 in rabbits during various stages of active infection and convalescence.     

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λ_max = 730 nm) and inactive Pr (λ_max = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB^Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyB^Ser86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.     

A kinetic study on the reductive opening of the diphenylmethylene acetal in methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside

Reductive opening of the diphenylmethyl acetal in methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside has been investigated by kinetic studies, and the results have been compared to those recently obtained by quantum chemical calculations. In contrast to the previous theoretical calculations which related only to the presumably rate limiting step of the reductive opening, the reaction system LiAlH₄, AlCl₃, and the title compound consists of at least four simultaneous reactions. Nevertheless, reasonable agreement can be found between the activation Gibbs free energy obtained from kinetic measurements and the theoretically calculated ones in spite of the experimental errors and the approximate nature of theoretical calculations.     

Functions of ABC transporters in plants

ABC (ATP-binding cassette) proteins are ubiquitously found in prokaryotes and eukaryotes and generally serve as membrane-intrinsic primary active pumps. In higher plants, ABC proteins constitute a large family, grouped phylogenetically into eight clusters, subfamilies ABCA-ABCI (ABCH is not found in plants). ABC transporters shuttle substrates as diverse as lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites and xenobiotic conjugates across a variety of biological membranes. To date, the largest proportions of characterized members have been localized to the plasma membrane and the tonoplast, with dominant implications in cellular secretion and vacuolar sequestration, but they are also found in mitochondrial, plastidal and peroxisomal membranes. Originally identified as tonoplast-intrinsic proteins that shuttle xenobiotic conjugates from the cytosol into the vacuole, thus being an integral part of the detoxification machinery, ABC transporters are now recognized to participate in a multitude of physiological processes that allow the plant to adapt to changing environments and cope with biotic and abiotic stresses.     

Obesity, diabetes mellitus, and liver fibrosis

Obesity is a global epidemic with more than 1 billion overweight adults and at least 300 million obese patients worldwide. Diabetes is characterized by a defect in insulin secretion or a decrease in sensitivity to insulin, which results in elevated fasting blood glucose. Both obesity and elevated fasting glucose are risk factors for nonalcoholic fatty liver disease, a disease spectrum that includes hepatic steatosis (nonalcoholic fatty liver), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Increased adiposity and insulin resistance contribute to the progression from NASH to fibrosis through the development of a profibrotic mileau in the liver, including increased hepatocellular death, increased reactive oxygen species generation, and an altered adipokine/cytokine balance. This review will summarize recent advances in our understanding of the pathological interactions among excessive fat accumulation, insulin resistance, and hepatic fibrogenesis and discuss specific molecular pathways that may be of interest in the development of therapeutic interventions to prevent and/or reverse hepatic fibrosis.     

In vitro shoot proliferation of Telekia speciosa (Schreb.) Baumg. Induced by different cytokinins

In vitro shoot multiplication of Telekia speciosa (Schreb.) Baumg. was tested on media containing benzyladenine, benzyladenine riboside, kinetin, zeatin, meta-topolin or 2-isopentenyladenine in different concentrations. We observed the proliferation rate, the length of shoots, rate of callus formation, and the presence of the hyperhydricity. The highest proliferation rate (13.17) was obtained on medium supplemented with 5.0 μM benzyladenine, however, the leaves were hyperhydrated at this concentration of benzyladenine, therefore for shoot multiplication lower (1.0 μM BA) concentration of benzyladenine is suggested. The longest shoots were achieved using 1.0 μM 2-iP. At this treatment 100% rooting was found, therefore the stage of rooting is omissible using 1.0 μM 2-iP during the multiplication. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant species.