Feasibility study of gel filtration as a separation method for blood serum proteins in combination with PIXE analysis

The combination between a protein separation technique and the PIXE method has a great potential for large surveys, including thousands of samples, in which multielemental analysis is required. Gel filtration with a Sephadex G-200 gel and a TRIS-acetate buffer was used for separating proteins in human serum. The fractions were doped with an yttrium/vanadium standard and then concentrated and pipeted onto Kimfol^? backing foils. Using the PIXE technique, the distributions of Fe, Cu, Zn, and Se, with respect to molecular size, were found, indicating binding to specific proteins. Sulfur and phosphorus were found to correlate well with the protein content measured by UV-absorption at 280 nm. Further developments and tests on the protein separation technique is required, taking restrictions imposed by the PIXE method into consideration.     

Microbial community composition and dynamics in high-temperature biogas reactors using industrial bioethanol waste as substrate

Stillage, which is generated during bioethanol production, constitutes a promising substrate for biogas production within the scope of an integrated biorefinery concept. In this study, a microbial community was grown on thin stillage as mono-substrate in a continuous stirred tank reactor (CSTR) at a constant temperature of 55 °C, at an organic loading rate of 1.5 g_oTS/L*d and a retention time of 25 days. Using an amplicon-based dataset of 17,400 high-quality sequences of 16S rRNA gene fragments (V2–V3 regions), predominance of Bacteria assigned to the families Thermotogaceae and Elusimicrobiaceae was detected. Dominant members of methane-producing Euryarchaeota within the CSTR belonged to obligate acetoclastic Methanosaetaceae and hydrogenotrophic Methanobacteriaceae. In order to investigate population dynamics during reactor acidification, the organic loading rate was increased abruptly, which resulted in an elevated concentration of volatile fatty acids. Acidification led to a decrease in relative abundance of Bacteria accompanied with stable numbers of Archaea. Nevertheless, the abundance of Methanosaetaceae increased while that of Methanobacteriales decreased successively. These findings demonstrate that a profound intervention to the biogas process may result in persistent community changes and reveals uncommon bacterial families as process-relevant microorganisms.     

Effects of high glucose and high insulin concentrations on osteoblast function in vitro

Bone disease as a consequence of diabetes mellitus (DM) is not fully understood. The effects of high glucose (30 mM), high insulin (50 nM), or mannitol (30 mM; osmotic control) were evaluated on MC3T3-E1 cells (osteoblasts) in vitro. The mRNA and protein levels of parathyroid hormone (PTH) receptor (PTH1R), collagen I, RANKL, osteoprotegerin (OPG), alkaline phosphatase (ALP), and glucose transporter (GLUT1) were estimated by real-time polymerase chain reaction or Western blotting. The mineralization capacity was analyzed by von Kossa staining. High glucose induced overexpression of RANKL (2×) and OPG (30×), suggesting that RANKL-induced osteoclast activity might not be a dominant mechanism of bone disease in DM, since this increase was followed by increased OPG. Collagen I increased by 12×, indicating an excess of organic matrix production. The expression of ALP decreased by 50 %, indicating a deficit in mineralization capacity, confirmed by von Kossa staining. Mannitol induced similar effects as glucose suggesting that extracellular hyperosmolarity was able to stimulate organic matrix production. GLUT1 expression was not altered, and insulin did not reverse most of the effects of glucose, suggesting that glucose uptake by osteoblasts was not altered by high glucose. The data suggest that the bone fragility typical of DM is not a consequence of excessive bone reabsorption but is instead attributable to a defect in organic matrix mineralization. The heightened increase in OPG versus RANKL might cause a decrease in the bone-remodeling cycle. Osteoblasts appear to be more sensitive to extracellular hypertonicity than to the intracellular metabolic effects of hyperglycemia.     

The influence of EDTA on the composition of alginate synthesized by Azotobacter vinelandii

1. During an investigation of the physiology of Azotobacter vinelandii with particular reference to polysaccharide formation, a suitable medium which was precipitate-free was developed by adding EDTA at a concentration of 50 mg/l to a basal medium containing one of eight different carbohydrates as sole carbon source.
2. Acetylated alginate was always produced by the organism when cultured under defined conditions, regardless of the carbohydrate source incorporated in the basal medium.
3. When EDTA was added to the medium, the bacteria produced acetylated polyuronides with a preponderance of mannuronic acid residues.
4. A comparison of the infrared spectra of the alginate produced by Azotobacter vinelandii and the affect of EDTA upon the mannuronic acid/guluronic acid ratios of the alginate are reported.

     

Reversal of cytogenetic action of 5-fluordeoxyuridine in barley root meristems by thymidine

The reversal of the cytogenetic effects of 5-fluordeoxyuridine, which are the gradual decrease of the mitotic index and the induction of chromosomal fragments was followed after addition of different concentrations of thymidine. Thymidine in the concentration 10⁴ M reversed both the effects of FUdR completely. The concentration of thymidine 10⁵ M partly reversed both the decrease of the mitotic index and the induction of chromosomal fragments. Uridine also caused some decrease of the cytogenetic effects of 5-fluordeoxyuridine.     

Microbial attachment to particles in marine and freshwater ecosystems

Scanning electron microscopy observations ofin situ suspended marine and freshwater particles show diverse but similar modes of bacterial and fungal attachment. A survey of Sierra Nevada mountain lakes and pelagic and near-shore waters in the Pacific Ocean indicates that attachment is most noticeable in the near-surface waters where fresh dissolved and particulate input of carbon from phytoplankton and elevated temperatures favor microbial growth. The most common modes of attachment are: adhesive stalk formation, growth on adhesive webs, attachment by the use of pili-like appendages and slimy capsular secretions, and molecular or chemical sorption without the use of visualized structural appendages. Attached microbial growth is accelerated when particulate substrates are supplied, even when they are not rich in organic nutrients. This is the case in the Lake Tahoe basin, where microflora attached to eroded silts can significantly modify the organic carbon and nutrient content of such minerogenous particles.     

Co-pigmentation and the color change with age in petals ofFuchsia hybrida

Pure or genuine malvin was isolated fromFuchsia petals for the first time and characterized as the malvin anhydro-base. The conditions for the co-pigmentation were examinedin vitro with regard to anthocyanin, co-pigment and pH, and the co-pigmentation occurred as the result of interaction between anthocyanin and co-pigment without any participation of metallic elements. The blue-violet color of youngFuchsia petals appeared at pH 4.8 in the 1∶0.6 molar ratio of anthocyanin to co-pigments. The color change from blue-violet in young petals to purple-red in old ones was caused by co-pigmentation and the pH change from 4.8 to 4.2. The decrease of pH in the old petals was due to the increase of organic acids such as aspartic, malic and tartaric acids.     

Development of an immunoenzyme method for detection and quantitative determination of microcystins

An indirect competitive immunoenzyme method for the quantitative estimation of microcystins (MCs) in water (MC-ELISA) using prepared MC-specific polyclonal antibodies was developed. The threshold concentration of the most widespread and highly toxic MC-LR, which was reliably detected using MC-ELISA, was 0.05 ± 0.01 ng/mL; the 50% inhibition concentration was 0.41 ± 0.05 ng/mL; and the concentration range for the quantitative estimation of MC-LR was 0.1–5.0 ng/mL. The MC-ELISA made it possible to detect MC-LR in water at concentrations 10–20 times lower than the World Health Organization guideline level for drinking water and 100–200 times lower than the allowable MC concentrations in water bodies. A group of cross-reacting MCs and nodularin was detected using MC-ELISA. This method can be applied for monitoring MC concentrations in water bodies and drinking water.     

Isolation and identification of Paenibacillus sp. FM-6, involved in the biotransformation of albendazole

A strain, designated as FM-6, was isolated from fish. Based on the results of phenotypic, physiological characteristics, genotypic and phylogenetic analysis, strain FM-6 was finally identified as Paenibacillus sp. When albendazole was provided as the sole carbon source, strain FM-6 could grow and transform albendazole. About 82.7 % albendazole (50 mg/L) was transformed by strain FM-6 after 5 days incubation at 30 °C, 160 rpm. With HPLC–MS method, the transforming product of albendazole was researched. Based on the molecular weight and the retention time, product was identified as albendazole sulfoxide and the transforming pathway of albendazole by strain FM-6 was proposed finally. The optimum temperature and pH for the bacterium growth and albendazole transformation by strain FM-6 were both 30 °C and 7.0. Moreover, the optimum concentration of albendazole for the bacterium growth was 50 mg/L. Coupled with practical production, 50 mg/L was the optimum concentration of albendazole transformation for strain FM-6. This study highlights an important potential use of strain FM-6 for producing albendazole sulfoxide.     

Effects of different 2,4-D concentrations on the cytogenetic behaviour of plant cells culturedin vitro

Cytogenetic effects of different concentrations of 2,4-D on suspension cultures ofVicia hajastana Grossh. andHaplopappus gracilis (Nutt.) Gray were studied using a simple, chemically defined medium. InVicia, the frequency of dicentric chromosomes showed a negative association with the 2,4-D concentration. In both the species, the frequencies of anaphase bridges were negatively associated, while those of the diploid cells showed a positive association with the 2,4-D concentration in the culture medium. A decrease in the frequency of diploid cells was observed in the cultures transferred to a lower concentration of 2,4-D. This decrease was transient inVicia, while inH. gracilis a new equilibrium at a lower level was established. Both endoreduplication and selection appear to be involved in the achievement of the lower equilibrium inH. gracilis, although selection appears to be the major factor.     

Sepsis-induced changes in amino acid transporters and leucine signaling via mTOR in skeletal muscle

The present study tested the hypothesis that sepsis-induced leucine (Leu) resistance in skeletal muscle is associated with a down-regulation of amino acid transporters important in regulating Leu flux or an impairment in the formation of the Leu-sensitive mTOR–Ragulator complex. Sepsis in adult male rats decreased basal protein synthesis in gastrocnemius, associated with a reduction in mTOR activation as indicated by decreased 4E-BP1 and S6K1 phosphorylation. The ability of oral Leu to increase protein synthesis and mTOR kinase after 1 h was largely prevented in sepsis. Sepsis increased CAT1, LAT2 and SNAT2 mRNA content two- to fourfold, but only the protein content for CAT1 (20 % decrease) differed significantly. Conversely, sepsis decreased the proton-assisted amino acid transporter (PAT)-2 mRNA by 60 %, but without a coordinate change in PAT2 protein. There was no sepsis or Leu effect on the protein content for RagA-D, LAMTOR-1 and -2, raptor, Rheb or mTOR in muscle. The binding of mTOR, PRAS40 and RagC to raptor did not differ for control and septic muscle in the basal condition; however, the Leu-induced decrease in PRAS40·raptor and increase in RagC·raptor seen in control muscle was absent in sepsis. The intracellular Leu concentration was increased in septic muscle, compared to basal control conditions, and oral Leu further increased the intracellular Leu concentration similarly in both control and septic rats. Hence, while alterations in select amino acid transporters are not associated with development of sepsis-induced Leu resistance, the Leu-stimulated binding of raptor with RagC and the recruitment of mTOR/raptor to the endosome-lysosomal compartment may partially explain the inability of Leu to fully activate mTOR and muscle protein synthesis.     

Action of Ypenyl [5-bis-(2-chloroethyl)-aminomethyluracil] on the chromosomes ofVicia faba

An investigation was carried out on ability of Ypenyl,i.e. 5-bis-(2-chloroethyl)-aminomethyluracil to induce structural disturbances of chromosomes and changes of mitotic activity of meristematic cells ofVicia faba. The cytostatic employed induced chromosomal aberrations mostly of the chromatide type, the greater part of which were localized in the group of small chromosomes. The frequency of aberrations was proportional to the concentration of the substance used as well as to the duration of treatment. After a 24 hour treatment with 0·01mm solution of Ypenyl the maximum incidence of aberrations was found after 8 hour recovery in water. Longer recovery periods resulted in a gradual decrease in the number of aberrant post-metaphases in the cell population studied. Increasing concentration of Ypenyl led to the decrease in mitotic activity. Short recovery periods caused a considerable decrease in the mitotic index, which was compensated as late as 16 hours after treatment with the cytostatic.     

Evidence for the in vivo regulation of glucose 6-phosphate dehydrogenase activity in Hydrogenomonas eutropha H 16 from measurements of the intracellular concentrations of metabolic intermediates

The inhibition of fructose utilization by whole cells of Hydrogenomonas eutropha H 16, following the addition of hydrogen to the gas phase, has been explained as an inhibition of glucose 6-phosphate dehydrogenase (Blackkolb and Schlegel, 1968a, b). The intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, three inhibitors of the enzyme (NADH, ATP and phosphoenolpyruvate) and some related metabolites were measured in cells incubated in the presence and absence of hydrogen. Inhibition of glucose 6-phosphate dehydrogenase was confirmed by an increase in the glucose 6-phosphate pool and a decrease in the 6-phosphogluconate concentration. The regulatory control is apparently due to a threefold increase in the NADH concentration while the concentrations of the other two inhibitors fell slightly. When the measured intracellular concentrations of intermediates were used in the in vitro assay of glucose 6-phosphate dehydrogenase activity, an almost total inhibition of the dehydrogenase was observed, therefore further regulatory factors must be considered.     

Inhibitory Action of Pig Alpha-Fetoprotein Early Activated Thymocyte Surface trohugh Na+,K+-Adenosinetriphosphatase

An immunomodulatory effect of pig alpha-fetoprotein (AFP) on early phytohaemagglutinin (PHA)-stimulated intact thymocytes from piglets was analyzed. The activity of surface-localized Na⁺, K⁺-adenosinetriphosphata sewas estimated, as the early reaction of mitogen-stimulated T-lymphocytes. A high inhibition of PHA-stimulated enzyme activity was found in the presence of AFP. The reaction was dependent on PHA concentration when using a constant AFP concentration. The highest AFP-induced decrease in PHA-induced Na⁺, K⁺-ATPase activity was 72 %. AFP was isolated by a new technique based on salting out in ammonium sulphate and acetate buffer solutions. A gentle, simple and rapid procedure makes it possible to obtain AFP in a high degree of purity with preserved biological activity. The presented results can contribute to understanding the molecular aspects of AFP immunoregulation of lymphocyte reactivityin vitro.     

The Effects of Individual Upper Alpha Neurofeedback in ADHD: An Open-Label Pilot Study

Standardized neurofeedback (NF) protocols have been extensively evaluated in attention-deficit/hyperactivity disorder (ADHD). However, such protocols do not account for the large EEG heterogeneity in ADHD. Thus, individualized approaches have been suggested to improve the clinical outcome. In this direction, an open-label pilot study was designed to evaluate a NF protocol of relative upper alpha power enhancement in fronto-central sites. Upper alpha band was individually determined using the alpha peak frequency as an anchor point. 20 ADHD children underwent 18 training sessions. Clinical and neurophysiological variables were measured pre- and post-training. EEG was recorded pre- and post-training, and pre- and post-training trials within each session, in both eyes closed resting state and eyes open task-related activity. A power EEG analysis assessed long-term and within-session effects, in the trained parameter and in all the sensors in the (1–30) Hz spectral range. Learning curves over sessions were assessed as well. Parents rated a clinical improvement in children regarding inattention and hyperactivity/impulsivity. Neurophysiological tests showed an improvement in working memory, concentration and impulsivity (decreased number of commission errors in a continuous performance test). Relative and absolute upper alpha power showed long-term enhancement in task-related activity, and a positive learning curve over sessions. The analysis of within-session effects showed a power decrease (“rebound” effect) in task-related activity, with no significant effects during training trials. We conclude that the enhancement of the individual upper alpha power is effective in improving several measures of clinical outcome and cognitive performance in ADHD. This is the first NF study evaluating such a protocol in ADHD. A controlled evaluation seems warranted due to the positive results obtained in the current study.     

Ammonia volatilization during aerobic and anaerobic manure decomposition

Ammonia volatilization, nitrogen immobilization, carbon decomposition and formation of volatile fatty acids was investigated in a laboratory incubation experiment with fresh poultry manure, to which increasing amounts of straw were added. Less than 1% of the manure nitrogen was volatilized as ammonia during anaerobic decomposition due to low pH values. In aerobic manure alkaline conditions prevailed and between 9 to 44% of the nitrogen was volatilized as ammonia. The volatilization courses could be described by a parallel first-order model. Increasing straw additions reduced ammonia volatilization during aerobic decomposition. Straw caused no immobilization of nitrogen under anaerobic conditions. In aerobic manure, nitrogen was mainly bound in organic forms whereas in anaerobic manure about two-thirds of the nitrogen was in ammonium form. C/N ratios in the organic matter of anaerobic manure were higher (33.1–87.5) than in the aerobic manure (9.5–18.0).     

Modulation of aquaporin 2 expression in the kidney of young goats by changes in nitrogen intake

In ruminants, a decrease of dietary nitrogen (N) is an appropriate feeding concept to reduce environmental pollution and costs. In our previous study, when goats were kept on an N-reduced diet, a decrease of plasma urea concentration and an increase of renal urea transporters were demonstrated. Renal urea absorption plays a crucial role for renal water absorption and urine concentration. Renal collecting duct water absorption is mainly mediated by the water channel aquaporin 1 and 2 (AQP1 and AQP2). Therefore, the aim of the present study was to investigate the effects of a dietary N reduction on expression of renal AQP1 and AQP2 in young goats. Twenty male White Saanen goats, 3 months old, were divided equally into two feeding groups, receiving either a diet with an adequate or a reduced-N supply. Goats fed a reduced-N diet showed significantly higher amounts of AQP1 mRNA in cortical tissue, and the expression of AQP2 mRNA and protein were highly elevated in renal outer medulla. An increase of vasopressin concentrations in plasma were detected for the N-reduced fed goats. Therefore, a stimulation of renal water absorption can be assumed. This might be an advantage for ruminants in times of N reduction due to higher urea concentrations in the tubular fluid and which might result in higher absorption of urea by renal urea transporters. Therefore, interplay of aquaporin water channels and urea transporters in the kidney may occur to maintain urea metabolism in times of N scarcity in young goats.