Fructan Biosynthesis by Intra‐ and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra‐ and extracellular levansucrase produced by the Z. mobilis strain 113 “S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that the cell free culture liquid, containing ethanol, levan, gluconic acid and sucrose (15%) at 25 °C, did not show any additional levan synthesising activity. At optimum FOS synthesis conditions (45 °C and 70% sucrose), the cell‐free culture liquid exhibited a high FOS synthesising activity (31% from total carbohydrates), with slightly reduced biomass activity. It was concluded that as a result of the simultaneous ethanol and levan production, the remaining biomass as well as the cell‐free culture liquid could be used for FOS production.     

Influence of muscle glycogen availability on ERK1/2 and Akt signaling after resistance exercise in human skeletal muscle.

Two pathways that have been implicated for cellular growth and development in response to muscle contraction are the extracellular signal-regulated kinase (ERK1/2) and Akt signaling pathways. Although these pathways are readily stimulated after exercise, little is known about how nutritional status may affect stimulation of these pathways in response to resistance exercise in human skeletal muscle. To investigate this, experienced cyclists performed 30 repetitions of knee extension exercise at 70% of one repetition maximum after a low (2%) or high (77%) carbohydrate (LCHO or HCHO) diet, which resulted in low or high (approximately 174 or approximately 591 mmol/kg dry wt) preexercise muscle glycogen content. Muscle biopsies were taken from the vastus lateralis before, approximately 20 s after, and 10 min after exercise. ERK1/2 and p90 ribosomal S6 kinase phosphorylation increased (P < or = 0.05) 10 min after exercise, regardless of muscle glycogen availability. Akt phosphorylation was elevated (P < 0.05) 10 min after exercise in the HCHO trial but was unaffected after exercise in the LCHO trial. Mammalian target of rapamycin phosphorylation was similar to that of Akt during each trial; however, change or lack of change was not significant. In conclusion, the ERK1/2 pathway appears to be unaffected by muscle glycogen content. However, muscle glycogen availability appears to contribute to regulation of the Akt pathway, which may influence cellular growth and adaptation in response to resistance exercise in a low-glycogen state.     

Inducible heat shock protein 70 promotes myelin autoantigen presentation by the HLA class II

In this study, we investigated the role of the inducible form of heat shock protein 70 (hsp70) in the presentation of the major putative autoantigen in multiple sclerosis, myelin basic protein (MBP), in the context of appropriate MHC class II. By coimmunoprecipitation, we found that MBP is associated with hsp70 in APC in an ATP/ADP-dependent manner. Additionally, using confocal microscopy, hsp70 was detected in the endocytic pathway of APC, where it colocalized with MBP and HLA-DR. The immunodominant epitopes of MBP 85-99 and 80-99 were shown to bind selectively and specifically to hsp70 by surface plasmon resonance. The functional significance of MBP interaction with hsp70 was demonstrated by the detection of enhanced responses of an MBP-specific T cell hybridoma to MBP and MBP 80-99 with increasing levels of hsp70 and reduced responses when hsp70 expression was diminished within APC-expressing DRA*0101, DRB1*1501 (DR1501). However, when MBP 85-99 was used as the stimulus, T cell hybridoma responses were not enhanced by hsp70 overexpression within APC, suggesting that hsp70 contributes to Ag processing rather than Ag presentation. The importance of a direct association between MBP and hsp70 in the presentation pathways was demonstrated by enhanced efficacy of MBP presentation by APC transfected with a plasmid vector encoding a fusion hsp70-MBP protein. This is the first report on the involvement of self-inducible hsp70 in MHC class II-dependent autoantigen processing by APC. It implicates that aberrant self hsp expression may lead to the enhancement/modulation of autoimmune responses.     

Factors affecting determination of superoxide anion generated by mitochondria from barley roots after anaerobiosis

During the post-hypoxic period, symptoms of oxidative stress and activation of enzymatic and non-enzymatic antioxidant systems were observed in several plant tissues. In the roots, mitochondrial respiratory chain is the main source of ROS. Superoxide anion radical is formed in the mitochondrial electron-transport chain at the level of Complexes I and III. The purpose of this work was to estimate superoxide anion production by the mitochondria isolated after a period of hypoxic treatment. Seedlings of barley (Hordeum vulgare L.) were grown on a nutrient medium flushed for 5 d with air (control) or nitrogen (hypoxia) and then transferred for 24 h to aerated medium (post-hypoxia). Production of superoxide anion by the mitochondria was measured by SOD-inhibitable oxidation of adrenaline to adrenochrome with NADH as a respiratory substrate. Hypoxic treatment increased mitochondrial activity but decreased mitochondrial superoxide anion appearance outside the mitochondrial membrane as compared to the mitochondria isolated from the roots continuously grown on aerated medium. The result of lower superoxide anion determination is attributed to increased antioxidants concentration during hypoxia. This was confirmed by inhibition of O₂⁻ production by exogenous GSH and stimulation by addition of 1-chloro-2,4-dinitrobenzene (CDNB), which depleted endogenous mitochondrial GSH.     

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.     

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.     

Development in vitro of the neuromusculature of two strigeid trematodes,Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.     

Damage to erythrocytes caused by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (in vitro)

The effects of the exposure of human erythrocytes to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin were studied. Particular attention was paid to lipid peroxidation, haemoglobin oxidation, and changes in the activity of catalase and glutathione peroxidase. Human erythrocytes at a 5% haematocrit were incubated with 2,3,7,8-TCDD at concentrations of 0.2 ppm to 1.6 ppm (ng-microg/ml erythrocytes) for 1 hour. The results obtained show that 2,3,7,8-TCDD induces the generation of lipid peroxides and the oxidation of Hb, and decreases the activity of catalase and glutathione peroxidase. This supports the thesis that TCDD causes oxidative stress in erythrocytes.