A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).     

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-d-glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.     

Determination of fatty acid compositions and cholesterol levels of some freshwater fish living in Porsuk Dam,Turkey

We determined the oil content, fatty acid composition, and cholesterol content of common carp (Cyprinus carpio), crucian carp (Carassius carassius), chub (Leusiscus cephalus), and tench (Tinca tinca) by GLC. The saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) levels were found to be 36.49%, 31.92%, 31.59% in common carp; 32.92%, 32.21%, and 34.87% in crucian carp; 36.19%, 32.91%, and 30.90% in chub; and 32.86%, 30.77%, and 36.37% in tench, respectively. The cholesterol (mg/100 g oil) levels of common carp, crucian carp, chub, and tench were determined by GLC methods as 119 ± 2.64 mg, 170.37 ± 2.36 mg, 94.68 ± 3.13 mg, and 179.84 ± 6.75 mg, respectively. Thus, the cholesterol contents of the analyzed freshwater fish species were low but their PUFA contents and nutritional values were high. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 15–17, January–February, 2009.     

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).     

Cellulases and Xylanases Production by <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> Grown on Sugar Cane Bagasse in Solid-State Fermentation

To investigate the production of cellulases and xylanases from Penicillium echinulatum 9A02S1, solid-state fermentation (SSF) was performed by using different ratios of sugar cane bagasse (SCB) and wheat bran (WB). The greatest filter paper activity obtained was 45.82 ± 1.88 U gdm⁻¹ in a culture containing 6SCB/4WB on the third day. The greatest β-glucosidase activities were 40.13 ± 5.10 U gdm⁻¹ obtained on the third day for the 0SCB/10WB culture and 29.17 ± 1.06 U gdm⁻¹ for the 2SCB/8WB culture. For endoglucanase, the greatest activities were 290.47 ± 43.57 and 276.84 ± 15.47 U gdm⁻¹, for the culture 6SCB/4WB on the fourth and fifth days of cultivation, respectively. The greatest xylanase activities were found on the third day for the cultures 6SCB/4WB (36.38 ± 5.38 U gdm⁻¹) and 4SCB/6WB (37.87 ± 2.26 U gdm⁻¹). In conclusion, the results presented in this article showed that it was possible to obtain large amounts of cellulases and xylanases enzymes using low-cost substrates, such as SCB and WB.     

Molecular Cloning and Expression of Two Cytosolic Copper–Zinc Superoxide Dismutases Genes from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>

Two cytosolic copper–zinc superoxide dismutase (cytCuZnSOD) complementary deoxyribonucleic acid were achieved in Nelumbo nucifera (Elian). The active sites and common characteristics of cytCuZnSOD family were showed by homology modeling. The two recombinant proteins expressed by PET-32a vector showed the similar SOD activity (89.94 ± 0.54 U/mg) and could maintain more than 90% activity after incubation at 65°C. The subcellular location by green fluorescent protein revealed that these two isoforms were all located in cytosol and nucleus. The cytCuZnSODs were expressed in various parts of N. nucifera, which were expressed highest in the leafstalks and young leaves and lowest in the roots. The cytCuZnSOD messenger ribonucleic acids isolated from wounded leaves significantly increased at 1.5 h after treatment (HAT) with the highest expression at 3 HAT, after which the level decreased.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

<Emphasis Type="Italic">S</Emphasis>-glutathionyl quantification in the attomole range using glutaredoxin-3-catalyzed cysteine derivatization and capillary gel electrophoresis with laser-induced fluorescence detection

S-glutathionylation (Pr–SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer’s disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr–SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model–S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr–SSG at 232 ± 10.64 (average ±SD; n = 3) nmol/mg. In the AD–Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD–Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr–SSG detection at 1.8 attomole (amol) level, reported to date.     

Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on-line chemical analysis of cultured adipocyte secretions

A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from ∼6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 ± 2.4 μM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 × 10⁻⁵ nmol cell⁻¹ min⁻¹. Upon pharmacological treatment with a β-adrenergic agonist to stimulate lipolysis, a 6.9 ± 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.     

Expression of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MA139 β-mannanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and the Enzyme Characterization

The 1014 nucleotides long gene-encoding β-mannanase from Bacillus subtilis strain MA139 was cloned using PCR. To obtain high expression levels in Pichia pastoris, the β-mannanase gene was optimized according to the codon usage bias of P. pastoris and fused downstream of GAP promoter. The reconstituted plasmid pGAP-mann was transformed into P. pastoris X-33 strain to constitutively express β-mannanase. When cultured at 28 °C for 3 days protein yields up to 2.7 mg/mL was obtained with the enzyme activity of up to 230 U/mL. In comparison, wild-type gene product yielded 1.9 mg/mL and 170 U/mL, respectively indicating that the protein yield and enzyme activity were significantly improved by codon modification. After purification, the enzyme properties were characterized. The optimal activity was at pH 6.0 and 50 °C. In the pH range of 3.0 to 9.0, β-mannanase showed above 60% of its peak activity. Among the numerous ions tested copper significantly inhibited the enzyme activity. These results suggested that codon-optimized β-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.     

Preventive Effect of Low Molecular Weight Glycosaminoglycan from <Emphasis Type="Italic">Amussium pleuronectus</Emphasis> (Linne) on Oxidative Injury and Cellular Abnormalities in Isoproterenol-Induced Cardiotoxicity in Wistar Rats

The present work explores suspicious consequence of low molecular weight glycosaminoglycan (LMW-GAG) on oxidative stress and cellular abnormalities in isoproterenol (ISO)-induced myocardial infarction in an experimental model. Group-III male Wistar rats (140 ± 10 g) were administrated by ISO (85 mg ISO/ml subcutaneously (SC) injected at the last two days of a 2–week period). Group-IV rats were treated LMW-GAG plus ISO (300 μg/day per rat SC for 1 week followed by 85 mg/kg ISO on the end last two days of the 2 - weeks). Untreated control (Group-I) and LMW-GAG drug control (Group-II) were also included. Serum and tissue lactate dehydrogenase, aminotransferases, and creatine kinase activities were increased in ISO group, which were normalized by LMW-GAG pretreatment rats. Antioxidant enzymes – superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities and non-enzymatic enzyme reduced glutathione (GSH) were decreased in the ISO induced rats, and this was increased by LMW-GAG pretreatment. Increased level of thiobarbituric acid reactive substances (TBARS) in plasma and the heart of ISO treated rats; pre s.c. injected with LMW-GAG to ISO-induced rats decreased the levels of TBARS. Histological examination revealed that the ISO-induced deleterious changes in the heart tissues were offset by LMW-GAG treatment. LMW-GAG affords considerable protection to the tissues challenged by cardiotoxicity, evidenced by its correction and restoration of serum and tissue indices of injury, to normalcy.     

Application of inductively coupled plasma–mass spectrometry (ICP–MS) and quality assurance to study the incorporation of strontium into bone,bone marrow,and teeth of dogs after one month of treatment with strontium malonate

The strontium content of serum, bone, marrow, and teeth was determined by inductively-coupled plasma mass spectrometry (ICP–MS). Significant correlations were obtained after the data were subjected to quality assurance (QA) performed according to validated procedures. After four weeks of treatment with strontium malonate, strontium levels increased from 76 ± 9 μg g⁻¹ in placebo-treated dogs to levels of 7.2 ± 1.7 mg g⁻¹, 9.5 ± 2.7 mg g⁻¹, and 9.8 ± 2.7 mg g⁻¹ in groups treated with 300, 1000, and 3000 mg kg⁻¹ day⁻¹, respectively. Strontium induced a highly significant increase in the bone formation marker, bone-specific alkaline phosphatase (BSAP), and an excellent correlation was found with the bone-strontium content. In females, the placebo-treated group showed a decrease in BSAP of 53%, whereas the three strontium malonate-treated groups showed an increase of 60, 276, and 278% for the groups treated with 300, 1000, and 3000 mg kg⁻¹ day⁻¹, respectively. For males the corresponding values were −44%, +142%, +194%, and +247% increases in BSAP in the placebo, 300, 1000, and 3000 mg kg⁻¹ day⁻¹ groups respectively.     

Banana Peel: A Potential Substrate for Laccase Production by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> VkJ2.4.5 in Solid-State Fermentation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l⁻¹) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l⁻¹) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min⁻¹. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.     

Silk-Fiber Immobilized Lipase-Catalyzed Hydrolysis of Emulsified Sunflower Oil

Lipase was immobilized in silk fibers through glutaraldehyde cross-linking to a maximum loading of 59 U/g silk-fiber and the immobilized lipase was utilized for the hydrolysis of sunflower oil (Helianthus annuus). The hydrolytic activity of the lipase, which was poor in biphasic oil in water system, was increased significantly when the sunflower oil was emulsified in aqueous medium. The hydrolytic activities of the immobilized lipase were 48.73 ± 1.26 U, 36.11 ± 0.96 U, and nil when the substrate sunflower oil was used as emulsion created by a rhamnolipid biosurfactant, Triton X100, and ultrasonication, respectively. Although the efficiency of the immobilized lipase was less than 12% than the corresponding free lipase, the immobilized lipase could be reused for the biosurfactant-mediated hydrolysis of sunflower oil up to third cycle of the reaction. The yield of the fatty acids in the second, third, and fourth cycles were 49.45%, 22.91%, and 5.09%, respectively, of the yield obtained in the first cycle.     

Quantification of luminally released serotonin in rat proximal colon by capillary electrophoresis with laser-induced fluorescence detection

Serotonin (5-hydroxytryptamine, 5-HT) plays vital roles in regulating gastrointestinal functions. Thus, the detection of 5-HT in the gastrointestinal tract is of great importance for biomedical research, medical diagnosis, and pharmaceutical therapy. This paper presents a simple, sensitive, and fast method for the quantification of luminally released serotonin in the feces and tissues of the rat proximal colon by means of capillary electrophoresis with laser-induced fluorescence detection. 5-Carboxyfluorescein N-succinimidyl ester was used for precolumn derivatization of serotonin. The optimal separation and detection conditions were obtained with an electrophoretic buffer containing 60 mM borate (pH 8.90) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The serotonin concentrations in the feces and tissues of proximal colons were analyzed with this method, and the average values of serotonin in the feces samples were 1.951 ± 0.446 ng/mg (male) and 2.095 ± 0.533 ng/mg (female) and 1.397 ± 0.267 ng/mg in rat proximal colon tissues. The results demonstrate that this method can accurately determine luminally released 5-HT in rats.     

Screening and Selection of Marine Isolate for l-Glutaminase Production and Media Optimization Using Response Surface Methodology

The current work details the screening of about 400 marine isolates from various marine niches, from which one isolate was finally selected based on the productivity of glutaminase (71.23 U/l). Further, biochemical identification tests and 16S rRNA sequencing identified this isolate to be Providencia sp. This isolate was taken up for further media optimization studies by using one-factor-at-a-time approach and subsequently by response surface methodology. A face centered central composite design was employed to investigate the interactive effects of four variables, viz., concentrations of glucose, methionine, urea, and succinic acid on glutaminase production. A significant influence of urea on glutaminase production was noted. Response surface methodology showed that a medium containing (g/l) glucose 10.0, urea 5.15, methionine 3.5, succinic acid 6.0, ammonium sulfate 2.5, and yeast extract 6.0 to be optimum for the production of glutaminase. The applied methodology was validated using this optimized media and enzyme activity 119 ± 0.12 U/l and specific activity of 0.63 U/mg protein after 28 h of incubation at 25 °C was obtained.     

Simultaneous quantitative analysis of letrozole, its carbinol metabolite, and carbinol glucuronide in human plasma by LC-MS/MS

Letrozole is an efficient endocrine treatment of postmenopausal breast cancer, however, not all patients benefit from this treatment, and moreover, severe side-effects like arthralgia frequently lead to discontinuation. To better understand inter-individual variability in drug response and side-effects, plasma analysis of steady-state concentrations of letrozole and its major metabolites is crucial. We developed a rapid, sensitive, and specific method for the simultaneous quantification of letrozole and its metabolites 4,4′-(hydroxymethylene)dibenzonitrile (carbinol) and bis(4-cyanophenyl)methyl hexopyranosiduronic acid (carbinol-gluc) by UHPLC-ESI-MS/MS using in-house synthesized, stable isotope-labeled internal standards. Following solid-phase extraction in BondElut C18 96-well plates, the analytes were separated on a ZORBAX Eclipse XDB-C18 column (1.8 μm, 4.6 × 50 mm) with a gradient of acetonitrile in 0.1% acetic acid in water and detected on a triple quadrupole mass spectrometer with electrospray ionization in the multiple reaction monitoring mode. Lower limits of quantification were 20, 0.2, and 2 nM for letrozole, carbinol, and carbinol-gluc, respectively. The assay has been validated according to FDA guidance and applied to the analysis of 20 plasma samples of postmenopausal breast cancer patients treated with 2.5 mg of letrozole per day. Mean plasma levels (±SD) were 366 ± 173, 0.38 ± 0.09, and 34 ± 12 nM for letrozole, carbinol, and carbinol-gluc, respectively. Our rapid and sensitive mass spectrometry based method enables future pharmacokinetic investigations of letrozole outcome.     

Enhancement of Human γ-Interferon Production in Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Batch Cultivation

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD₆₀₀ = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.     

Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography–high resolution mass spectrometry in mucosal biopsies of the lower esophagus

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 ± 1.9 mg tissue and a mean adduct level of 6.13 ± 9.35 pmol HPB/mg DNA.     

Fifty-gigahertz Microwave Exposure Effect of Radiations on Rat Brain

The object of this study is to investigate the effects of 50-GHz microwave radiation on the brain of Wistar rats. Male rats of the Wistar strain were used in the study. Animals of 60-day age were divided into two groups—group 1, sham-exposed, and group 2, experimental (microwave-exposed). The rats were housed in a temperature-controlled room (25 °C) with constant humidity (40–50%) and received food and water ad libitum. During exposure, rats were placed in Plexiglas cages with drilled ventilation holes and kept in an anechoic chamber. The animals were exposed for 2 h a day for 45 days continuously at a power level of 0.86 μW/cm² with nominal specific absorption rate 8.0 × 10⁻⁴ w/kg. After the exposure period, the rats were killed and homogenized, and protein kinase C (PKC), DNA double-strand break, and antioxidant enzyme activity [superoxides dismutase (SOD), catalase, and glutathione peroxidase (GPx)] were estimated in the whole brain. Result shows that the chronic exposure to these radiations causes DNA double-strand break (head and tail length, intensity and tail migration) and a significant decrease in GPx and SOD activity (p = <0.05) in brain cells, whereas catalase activity shows significant increase in the exposed group of brain samples as compared with control (p = <0.001). In addition to these, PKC decreased significantly in whole brain and hippocampus (p < 0.05). All data are expressed as mean ± standard deviation. We conclude that these radiations can have a significant effect on the whole brain.