Comparative study of multi-enzyme production from typical agro-industrial residues and ultrasound-assisted extraction of crude enzyme in fermentation with Aspergillus japonicus PJ01.

Submerged fermentation (SmF) and solid-state fermentation (SSF) of Aspergillus japonicus PJ01 for multi-enzyme complexes (MEC) production were comparatively studied. The results showed that orange peel and wheat bran were the best substrates for MEC production in SmF and SSF, respectively. After 72 h of cultivation under SmF, the maximal pectinase, CMCase, and xylanase activities reached 2610, 85, and 335 U/gds (units/gram dry substrate), respectively; while after 72 h of cultivation under SSF, these three enzymes' activities reached 966, 58, and 1004 U/gds, respectively. Effects of ultrasound on extraction of crude enzymes from SSF medium were determined, the maximal activities of pectinase, CMCase, and xylanase increased to 1.20, 1.48, and 1.30-fold, respectively. Apparent different mycelia growths of SSF and SmF were observed by scanning electron microscopy; and different isoforms of the crude enzyme extracts from SSF and SmF were presented by zymogram analysis.

Selective brain responses to acute and chronic low-dose X-ray irradiation in males and females.

Radiation exposure is known to have profound effects on the brain, leading to precursor cell dysfunction and debilitating cognitive declines [Nat. Med. 8 (2002) 955]. Although a plethora of data exist on the effects of high radiation doses, the effects of low-dose irradiation, such as ones received during repetitive diagnostic and therapeutic exposures, are still under-investigated [Am. J. Otolaryngol. 23 (2002) 215; Proc. Natl. Acad. Sci. USA 97 (2000) 889; Curr. Opin. Neurol. 16 (2003) 129]. Furthermore, most studies of the biological effects of ionizing radiation have been performed using a single acute dose, while clinically and environmentally relevant exposures occur predominantly under chronic/repetitive conditions. Here, we have used a mouse model to compare the effects of chronic/repetitive and acute low-dose radiation (LDR) exposure (0.5Gy) to ionizing radiation on the brain in vivo. We examined the LDR effects on p42/44 MAPK (ERK1/ERK2), CaMKII, and AKT signaling-the interconnected pathways that have been previously shown to be crucial for neuronal survival upon irradiation. We report perturbations in ERK1/2, AKT, and CREB upon acute and chronic/repetitive low-dose exposure in the hippocampus and frontal cortex of mice. These studies were paralleled by the analysis of radiation effects on neurogenesis and cellular proliferation. Repetitive exposure had a much more pronounced effect on cellular signaling and neurogenesis than acute exposure. These results suggest that studies of single acute exposures might be limited in terms of their predictive value. We also present the first evidence of sex differences in radiation-induced signaling in the hippocampus and frontal cortex. We show the role of estrogens in brain radiation responses and discuss the implications of the observed changes.

Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages.

Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1-10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity.

The role of the ubiquitin/proteasome system in cellular responses to radiation.

In the last few years, the ubiquitin(Ub)/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded, damaged, and effete proteins. This review examines the role of the Ub/proteasome system in processes underlying the classical effects of irradiation on cells, such as radiation-induced gene expression, DNA repair and chromosome instability, oxidative damage, cell cycle arrest, and cell death. Furthermore, recent evidence suggests that the proteasome is a redox-sensitive target for ionizing radiation and other oxidative stress signals. In other words, the Ub/proteasome system may not simply be a passive player in radiation-induced responses, but may modulate them. The extent of the modulation will be influenced by the functional and structural diversity that is expressed by the system. Cell types vary in the Ub/proteasome structures they possess and the level at which they function, and this changes as they go from the normal to the cancerous condition. Cancer-related functional changes within the Ub/proteasome system may therefore present unique targets for cancer therapy, especially when targeting agents are used in combination with radio- or chemotherapy. The peptide boronic acid compound PS-341, which was designed to inhibit proteasome chymotryptic activity, is in clinical trials for the treatment of solid and hematogenous tumors. It has shown some efficacy on its own and in combination with chemotherapy. Preclinical studies have shown that PS-341 will also potentiate the cytotoxic effects of radiation therapy. In addition, other drugs in common clinical use have been shown to affect proteasome function, and their activities may be valuably reconsidered from this perspective.

The plasma membrane-associated NADH oxidase (ECTO-NOX) of mouse skin responds to blue light.

NADH oxidases of the external plasma membrane surface (ECTO-NOX proteins) are characterized by oscillations in activity with a regular period length of 24 min. Explants of mouse skin exhibit the oscillatory activity as estimated from the decrease in A(340) suggesting that individual ECTO-NOX molecules must somehow be induced to function synchronously. Transfer of explants of mouse skin from darkness to blue light (495 nm, 2 min, 50 micromol m(-1) s(-1)) resulted in initiation of a new activity maximum (entrainment) with a midpoint 36 min after light exposure followed by maxima every 24 min thereafter. Addition of melatonin resulted in a new maximum 24 min after melatonin addition. The findings suggest that the ECTO-NOX proteins play a central role in the entrainment of the biological clock both by light and by melatonin.

The plasma membrane-associated NADH oxidase of spinach leaves responds to blue light.

The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks is characterized by oscillations in activity with a regular period length of ca. 24 min. Within a single population of plants exposed to light at the same time, NOX activities of all plants function synchronously. Exposure of plants transferred from darkness to blue light (495 nm, 2 min, 50 micromoles m-2 s-1) resulted in a complex response pattern but with a new maximum in the rate of NOX activity 36 (24+12) min after illumination and then with maxima in the rate of NOX activity every 24 min thereafter. Transient maxima in NOX activity were observed as well after 9.3 + /- 1.4 and 20.7 +/- 2.1 min. The blue light response differed from the response to red (650 nm, 10 min, 50 micromoles m-2 s-1) or white light where activity maxima were initiated 12 min after the light exposure followed by maxima every 24 min thereafter. Green or yellow light was ineffective. The light response was independent of the time in the 24-min NOX cycle when the light was given. The net effects of blue and red light were ultimately the same with a new maximum in the rate of NOX activity at 12+24=36 min (and every 24 min thereafter), but the mechanisms appear to be distinct.

Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.

UV radiation is the most important environmental skin aggressor, causing cancer and other problems. This paper reports the use of oligonucleotide microarray technology to determine changes in gene expression in human keratinocytes after UVB treatment. Examination of the effects of different doses at different times after irradiation gave a global picture of the keratinocyte response to this type of insult. Five hundred thirty-nine regulated transcripts were found and organized into nine different clusters depending on behavior patterns. Classification of these genes into 23 functional categories revealed that several biological processes are globally affected by UVB. In addition to confirming a majority up-regulation of the transcripts related to the UV-specific inflammatory and stress responses, significant increases were seen in the expression of genes involved in basal transcription, splicing, and translation as well as in the proteasome-mediated degradation category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly downregulated. These results demonstrate the complexity of the transcriptional profile of the UVB response, describe several cellular processes previously not known to be affected by UV irradiation, and serve as a basis for the global characterization of UV-regulated genes and pathways.

Photic regulation of melatonin in rat retina and the role of proteasomal proteolysis.

Several investigations have shown that illumination at night reduces melatonin level in the mammalian pineal, but the effect of night illumination on the retina is not known. In this study retinas were cultured in a flow-through apparatus and then were exposed to light at ZT 18. Light exposure reduced melatonin levels to the daytime level within 30 min. The reduction of melatonin levels was due to a rapid decrease in the activity of the enzyme AA-NAT; AA-NAT mRNA levels were not affected by illumination. Pre-incubation with lactacystin (25 microM) prevented light-induced reduction of AA-NAT activity and melatonin levels. These results demonstrate that melatonin levels in the mammalian retina are affected by light exposure at night, via proteosomal proteolysis of AA-NAT.

Cell surface NADH oxidase activity of brine shrimp oscillates with a period of 25 min and is entrained by light.

Plants have a surface NADH oxidase that measures time by oscillating with a 24-min period. The period is synchronized by light. With plants, a new maximum is observed exactly 12 min after the beginning of the light exposure. These experiments were to determine if animals exhibited a cell surface NADH oxidase having a similar periodicity and to answer the question, does the periodicity in animals respond to light? Using brine shrimp as a model, the findings show that plants and animals exhibit similar oscillating NADH oxidase activity and that the periodicity in this invertebrate animal does respond to light. Brine shrimp were grown for two to three days and transferred to darkness for 45 min. After return to light for one min, NADH was added and measurements of NADH oxidation were recorded over 50 min. The brine shrimp exhibited a cell surface NADH oxidase that oscillated with a period of 25 min. After being subjected to light, the brine shrimp showed a new maximum in NADH oxidation between 12 to 13 min after the beginning of the light exposure and again at 37 min and at 25 min intervals thereafter. The findings demonstrate that the periodic oscillations in NADH oxidation of brine shrimp are light entrainable.

The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

Effect of radiation on the xanthine oxidoreductase system in the liver of mice.

The xanthine oxidoreductase system is one of the major sources of free radicals in many pathophysiological conditions. Since ionizing radiations cause cell damage and death, the xanthine oxidoreductase system may contribute to the detrimental effects in irradiated systems. Therefore, modulation of the xanthine oxidoreductase system by radiation has been examined in the present study. Female Swiss albino mice (7-8 weeks old) were irradiated with gamma rays (1-9 Gy) at a dose rate of 0.023 Gy s(-1) and the specific activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were determined in the liver of the animals. The mode and magnitude of change in the specific activities of XO and XDH were found to depend on radiation dose. At doses above 3 Gy, the specific activity of XO increased rapidly and continued to increase with increasing dose. However, the specific activity of XDH was decreased. These findings are suggestive of an inverse relationship between the activity of XO and XDH. The ratio of the activity of XDH to that of XO decreased with radiation dose. However, the total activity (XDH + XO) remained constant at all doses. These results indicate that XDH may be converted into XO. An intermediate form, D/O, appears to be transient in the process of conversion. The enhanced specific activity of XO may cause oxidative stress that contributes to the radiation damage and its persistence in the postirradiation period. Radiation-induced peroxidative damage determined in terms of the formation of TBARS and the change in the specific activity of lactate dehydrogenase support this possibility.

MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.

Light-dependent ATP synthesis in mitochondria.

Light-dependent ATP synthesis was studied in an illuminated suspension of rat liver mitochondria. The action of light was shown to lead to an increase in the ATP content in the absence of oxidisable substrates and in the presence of high (hundreds of microM) ADP concentrations in the medium. At a relatively low (50 microM) ADP concentration, efficient light-dependent phosphorylation was observed in the presence of alpha-ketoglutarate. Prolonged illumination stimulated ATP hydrolysis. Rotenone, antimycin, azide, dicyclohexylcarbodiimide, and oligomycin inhibited the light-dependent phosphorylation almost completely. The level of ATP decreased under the action of 2,4-dinitrophenol in the dark but was restored by high light intensities. Blue light, 436 nm, was most efficient to produce light-dependent phosphorylation. It is assumed that quanta of vibrational excitation formed in the course of vibrational relaxation and the internal conversion of photoexcited flavoproteins and cytochromes are transferred to the ATP-synthetase and "eject" ATP from the active center, thus shifting the enzymatic reaction to ATP production.

Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides.

Cytochrome b-561 of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH greater than 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of approximately 1 ms after the flash, followed by reduction with t 1/2 approximately 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH2 produced in the pool on flash excitation.

Two distinct quinone-modulated modes of antimycin-sensitive cytochrome b reduction in the cytochrome bc1 complex.

Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc1 complex perhaps involving a direct QB-Qc site interaction. This latter reaction is suppressed by occupancy of the Qc site, not only by antimycin but by ubiquinol and ubiquinone.

Electron transport in Paracoccus halodenitrificans and the role of ubiquinone.

The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm). When the membranes were extracted with n-pentane, NADH oxidase activity was lost. Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes. Succinate oxidation was not inhibited by dicoumarol or HQNO, but was affected by ultraviolet irradiation or n-pentane extraction. However, the addition of the ubiquinone fraction to the membranes extracted with n-pentane did not restore enzyme activity. These observations suggested that NADH and succinate were not oxidized through a common ubiquinone pool.

Radiation inactivation of oligomeric enzyme systems: theoretical considerations.

Radiation inactivation has been used as a tool to explore the size, structure, and function of soluble and membrane-bound enzymes. Enzyme systems consisting of a single functional unit yield a single exponential dependence of enzyme activity on radiation dose. Complex, nonexponential, inactivation curves suggest the presence of a multiunit enzyme system. A concave-upward inactivation curve suggests the presence of multiple independent functional units of distinct size and activity that do not interact with each other. An oligomeric enzyme, consisting of n identical subunits in equilibrium with monomers, can give simple exponential decay curves or more complex inactivation curves with various degrees of upward or downward concavity, depending upon the extent of oligomer-monomer equilibration among subunits after radiation, oligomer-to-monomer size and activity ratios, and multihit requirements for oligomer inactivation. For each of these possibilities, equations for the inactivation curves are derived, calculated numerically, and discussed in qualitative terms. A systematic approach to the evaluation of complex radiation inactivation curves is proposed and limitations of the radiation inactivation method are reviewed.