An effective technique for the processing of saliva for the analysis of leptin and adiponectin

The recovery of protein from saliva has been extensively investigated as a method to monitor health. The aim of this study was to compare filtration and centrifugation as two methods of saliva processing necessary for determining the levels of salivary leptin and adiponectin. Thirty-seven healthy patients (median age of 45 years; range 35–73) participated in the study. Unstimulated whole saliva was collected by a drooling technique. An aliquot was filtered using a Millex-Millipore^® (0.45 μm PVDF Dura Pore membrane) syringe and a second aliquot was centrifuged at 15 000 × g for 15 min at 4 °C. Leptin and adiponectin levels were analyzed using an ELISA kit for serum (RayBio^®, GA, USA) with minor modifications. Leptin and adiponectin levels following the filtration technique yielded comparable results with those after centrifugation. Correlation was observed between filtered and centrifuged salivary leptin levels ((r = 0.9155; 95% CI 0.8362–0.9573; p < 0.0001) with concordance correlation coefficient k 0.9114 (95% CI 0.8332–0.9539)). Less correlation was observed for adiponectin ((r = 0.5718; 95% CI 0.3041–0.7558; p = 0.0002) with concordance correlation coefficient k 0.5586 (95% CI 0.2977–0.7419)). Using a Bland–Altman plot, similar measurements for both adipocytokines were observed with mean difference within a 95% CI, and interpreted as no systematic differences between the two processing techniques. This study showed that filtration is an alternative saliva processing technique to retrieve supernatant for protein analysis. Filtered saliva yielded leptin and adiponectin concentrations comparable with those obtained from centrifuged saliva.     

Different types of fruit damages of three internal apple feeders diagnosed with mitochondrial molecular markers

Three tortricid pests, Grapholita dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura), are known as internal apple feeders in Korea. To identify young larvae, this study developed two types of molecular markers from their mitochondrial DNA (mtDNA) sequences. To this end, six different loci of mtDNA were sequenced in G. dimorpha: cytochrome oxidase subunit I (460 bp), cytochrome oxidase subunit II (446 bp), cytochrome b (308 bp), NADH dehydrogenase 3 (585 bp), NADH dehydrogenase 4 (ND4, 835 bp), and 16S rRNA (1300 bp). These sequences were compared with those of G. molesta and C. sasakii in order to develop PCR–RFLP and diagnostic primers. ND4 locus was selected to be used for developing a PCR–RFLP marker. ND4-Swa I digests showed two bands for G. dimorpha, one band for G. molesta, and three bands for C. sasakii. On the other hand, species-specific diagnostic PCR primers were developed using ND4 locus. These markers were then applied to diagnose larvae infesting apples to determine species-specific fruit damage patterns, in which G. dimorpha, G. molesta, and C. sasakii showed different feeding behaviors in terms of their main feeding sites in apple fruits.     

Expression of aromatase,androgen and estrogen receptors in peripheral target tissues in diabetes

Our previous studies have shown that diabetes in the male streptozotocin (STZ)-induced diabetic rat is characterized by a decrease in circulating testosterone and concomitant increase in estradiol levels. Interestingly, this increase in estradiol levels persists even after castration, suggesting extra-testicular origins of estradiol in diabetes. The aim of the present study was to examine whether other target organs of diabetes may be sources of estradiol. The study was performed in male Sprague–Dawley non-diabetic (ND), STZ-induced diabetic (D) and STZ-induced diabetic castrated (Dcas) rats (n = 8–9/group). 14 weeks of diabetes was associated with decreased testicular (ND, 26.3 ± 4.19; D, 18.4 ± 1.54; P < 0.05), but increased renal (ND, 1.83 ± 0.92; D, 7.85 ± 1.38; P < 0.05) and ocular (D, 23.4 ± 3.66; D, 87.1 ± 28.1; P < 0.05) aromatase activity. This increase in renal (Dcas, 6.30 ± 1.25) and ocular (Dcas, 62.7 ± 11.9) aromatase activity persisted after castration. The diabetic kidney also had increased levels of tissue estrogen (ND, 0.31 ± 0.01; D, 0.51 ± 0.11; Dcas, 0.45 ± 0.08) as well as estrogen receptor alpha protein expression (ND, 0.63 ± 0.09; D, 1.62 ± 0.28; Dcas, 1.38 ± 0.20). These data suggest that in male STZ-induced diabetic rats, tissues other than the testis may become sources of estradiol. In particular, the diabetic kidney appears to produce estradiol following castration, a state that is associated with a high degree or renal injury. Overall, our data provides evidence for the extra-testicular source of estradiol that in males, through an intracrine mechanism, may contribute to the development and/or progression of end-organ damage associated with diabetes.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.     

Effects of urea and aqueous ammonia treatment on the composition and nutritive value of maize residues

Two trials were conducted to investigate changes in chemical composition and nutritive value of maize residues treated with urea and aqueous ammonia. In Experiment I, maize stalks, husks and cobs were chopped into pieces of approximately 1 cm length. Aqueous NH₃ (3%) and feed grade urea equivalent to 3% NH₃ were sprayed into 100 g duplicate samples on DM basis, thoroughly mixed, stored in plastic containers and kept for 3 week at ambient temperature (25–27 °C). Proximate composition and in vitro dry matter digestibility of test materials were determined. In Experiment II, 100 kg batches of the maize residues were chopped and treated as in Experiment I but stored in 205 l drums lined with black polythene sheet. Feed intake and nutrient digestibility of treated materials were evaluated with 18 mature WAD sheep averaging 19.8 kg BW. Treatment improved (P < 0.05) intake and digestion coefficients for N, DM, NDF, ADF and OM but there were no differences (P > 0.05) between aqueous NH₃ and urea treatments. Feed grade urea or the equivalent weight of fertilizer grade urea can be used to improve the nutritional value of maize residues for small ruminant feeding during off season periods.     

The crystallographic structure of Panicum Mosaic Virus (PMV)

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2₁ with a = 411.7 Å, b = 403.9 Å and c = 412.5 Å, with β = 89.7°. The asymmetric unit was two entire T = 3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ～20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca⁺⁺ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1–21 of the A, B and C subunits, respectively, and the four C-terminal residues (239–242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Analysis of growth patterns in purebred Kambing Katjang goat and its crosses with the German Fawn

The objective of this study was to investigate growth patterns of goats utilizing data from a crossbreeding program involving the exotic German Fawn (GF) and the indigenous Kambing Katjang (KK) goats. Growth curve models and growth curve parameters were compared and analyzed for different genotypes and litter types. A total of 20,393 weight–age data from 208 female goats belonging to various crossbreeding genotypes were individually fitted to four growth curve models (Brody, Bertalanffy, Gompertz and Logistic). The goodness of fit was highest in the Brody model in most cases. A comparison of R² among genotypes showed that they were highest for KK. There were no significant differences of genotypes for estimated mature weight in the Brody model. The estimated mature weights for KK were significantly lower (P < 0.05) than for GF × KK (F₁), backcrosses with 75% GF genes (BC) and F₁ × F₁ (F₂) in the other models. The correlations between estimated mature weights and the maturing rates were lowest for BC. The genotype significantly (P < 0.01) affected the age at the constant degree of maturity (67% and 90% of mature weight) in all models. The BC genotype was the youngest at maturity and KK the oldest. All models well expressed the growth pattern of the target animals when they were older than 2.5 years of age. The results from the present study showed that the growth pattern may be altered by crossbreeding of KK with the GF breed.     

Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from cultured Cordyceps militaris

A novel polysaccharide named CBP-1 was isolated from the fruiting body of cultured Cordyceps militaris by alkaline extraction as well as anion-exchange and gel-permeation chromatography. Its structural features were investigated by a combination of chemical and instrumental analysis approaches, including partial hydrolysis, methylation analysis, HIO₄ oxidation-Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD and FT-IR. The results indicated that CBP-1 has a backbone of (1 → 4)-α-d-mannose residues which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-α-d-glucose residues and (1 → 6)-β-d-galactose residues, and terminated with β-d-galactose residues. In the in vitro antioxidant assay, CBP-1 was found to possess the hydroxyl radical-scavenging activity with an IC₅₀ value of 0.638 mg/ml.     

Neuromuscular and psychological influences on range of motion recovery in anterior cruciate ligament reconstruction patients

To identify distinguishing characteristics for knee surgery patients who experience a protracted recovery process, we sought to determine if there is an association between the neuromuscular stretch reflex and psychological factors of pain perception and anxiety on the range of motion (ROM) recovery rate of post-operative anterior cruciate ligament reconstruction (ACLR) rehabilitation patients. The ACLR participants were categorized into a slow recovery group (SRG: >6 weeks to recover 0–125° knee flexion [n = 10]) and a normal recovery group (NRG: <6 weeks to recovery 0–125° knee flexion [n = 12]). Control participants (n = 22) were age, gender and activity-level matched to the surgical participants. Neuromuscular testing consisted of sagittal plane video kinematics of the Wartenberg Pendulum Test for determining lower limb stiffness indices and electromyography-monitored patellar tendon tap reflex responses. Psychological and health status assessments consisted of the State–Trait Anxiety Inventory and SF-36? Health Survey. Data revealed that neuromuscular reflex profiles, lower limb stiffness indices, pain, anxiety and SF-36? indices of function were not significantly different between the two surgical groups (SRG and NRG). The surgical groups exhibited significantly greater pain (2.67 ± 2.27 SRG, 1.49 ± 1.15 NRG) than the control group (p ? .05). SF-36? indices were significantly lower for the surgical groups for total score (546.55 ± 94.70 SRG, 577.57 ± 125.58 NRG), function 69.00 ± 20.24 SRG, 67.08 ± 19.12 NRG), role physical (53.75 ± 22.85 SRG, 53.12 ± 23.15 NRG), social (76.24 ± 25.31 SRG, 65.62 ± 27.24 NRG), and emotional (82.49 ± 19.81 SRG, 81.38 ± 23.02 NRG) subscales (p ? .05). These results suggest that neuromuscular reflex responses, visual analogue scale (VAS) pain, and anxiety are not distinguishing factors for ROM recovery rate between the SRG and NRG. Decreased SF-36? indices, including pain as it influences function, though clinically relevant factors, were not statistically associated with post-operative ROM recovery rate.     

Decreased lower limb muscle recruitment contributes to the inability of older adults to recover with a single step following a forward loss of balance

In response to a balance disturbance, older individuals often require multiple steps to prevent a fall. Reliance on multiple steps to recover balance is predictive of a future fall, so studies should determine the mechanisms underlying differences between older adults who can and cannot recover balance with a single step. This study compared neural activation parameters of the major leg muscles during balance recovery from a sudden forward loss of balance in older individuals capable of recovering with a single step and those who required multiple steps to regain balance. Eighty-one healthy, community dwelling adults aged 70 ± 3 participated. Loss of balance was induced by releasing participants from a static forward lean. Participants performed four trials at three initial lean magnitudes and were subsequently classified as single or multiple steppers. Although step length was shorter in multiple compared to single steppers (F = 9.64; p = 0.02), no significant differences were found between groups in EMG onset time in the step limb muscles (F = 0.033–0.769; p = 0.478–0.967). However, peak EMG normalised to values obtained during maximal voluntary contraction was significantly higher in single steppers in 6 of the 7 stepping limb muscles (F = 1.054–4.167; p = 0.045–0.024). These data suggest that compared to multiple steppers, single steppers recruit a larger proportion of the available motor unit pool during balance recovery. Thus, modulation of EMG amplitude plays a larger role in balance recovery than EMG timing in this context.     

Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.     

Effects of vitreousness and particle size of maize grain on ruminal and intestinal in sacco degradation of dry matter,starch and nitrogen

We assessed the effects of vitreousness and particle size of maize grain on ruminal and intestinal in sacco degradation of dry matter, starch and nitrogen. Six maize grain (Zea mays) genotypes characterized by differing vitreousness (proportion of vitreous in total endosperm) were ground (3-mm screen; Gr, ground particles, mean particle size (MPS): 526 μm) and cracked with a roller mill using two gap width settings (CS, cracked small particles, MPS: 1360 μm; CL, cracked large particles, MPS: 2380 μm). The ruminal and intestinal in sacco degradation of dry matter, starch and nitrogen was measured on three dry Holstein cows, fitted with rumen, proximal duodenum and terminal ileum cannulas, fed maize silage ad libitum twice daily. The ruminal starch degradability and intestinal digestibility differed among genotypes (P<0.001) and decreased as particle size increased (P<0.001). For the same particle size, starch ruminal degradability decreased (P<0.05) and intestinal digestibility decreased (P<0.002) with vitreousness. Particle size and vitreousness of maize grain are efficient factors for manipulating the amount of starch escaping rumen degradation, but may be limiting for the amount of starch digested in the small intestine.     

Fast biodegradation of long chain n-alkanes and crude oil at high concentrations with Rhodococcus sp. Moj-3449

Biodegradation of long chain n-alkanes and crude oil with fast rate and high concentration are desirable for bioremediation, especially in heavily oil-polluted areas, and enhanced oil recovery. We discovered Rhodococcus sp. Moj-3449 with such unique abilities by screening microorganisms for the growth on n-hexadecane at 30 mg/mL. The new strain grew very fast on 120 mg/mL of n-hexadecane giving a cell density of 14.7 g cdw/L after only 2 days’ incubation. During the growth with this strain, the oil–water phases were rapidly emulsified, giving rise to tolerance to high alkane concentration (250 mg/mL) and fast growth rate of 0.10–0.20 h^?1 for alkane concentration of 1–180 mg/mL. The degraded concentration of n-hexadecane increased linearly with the initial alkane concentration (1–250 mg/mL). Incubation on n-hexadecane at 250 mg/mL for 7 days gave a cell density of 13.5 g cdw/L and degraded 124 mg/mL of n-hexadecane. The strain grew also fast on n-dodecane (C12), n-tetradecane (C14), and n-octadecane (C18), with degradation preference of C14 (=C16) > C12 > C18. Different from many alkane-degrading strains, Rhodococcus sp. Moj-3449 was found to have subterminal oxidation pathway. Rhodococcus sp. Moj-3449 degraded also crude oil fast at 60–250 mg/mL, with a wide range of n-alkanes (C10–C35) as substrates in which C14–C19 are preferred. The degradation ability increased with initial oil concentration from 60 to 150 mg/mL and slightly decreased afterwards. Incubation on 150 mg/mL of crude oil for 7 days degraded 37% of n-alkanes. The outstanding ability of rapidly degrading long chain n-alkanes and crude oil at high concentration makes Rhodococcus sp. Moj-3449 potentially useful for bioremediation and microbial enhanced oil recovery.     

Synthesis and antimycobacterial evaluation of pyrazinamide derivatives with benzylamino substitution

A series of 19 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro whole cell antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two types of Mycobacterium avium. The series consisted of 3-(benzylamino)-5-cyanopyrazine-2-carboxamides and 3-(benzylamino)pyrazine-2,5-dicarbonitriles with various substituents on the phenyl ring. RP-HPLC method was used to determine the lipophilicity of the prepared compounds. Nine compounds exerted similar or better activity against Mycobacterium tuberculosis compared to pyrazinamide (MIC = 6.25–12.5 μg/mL). 3-(Benzylamino)pyrazine-2,5-dicarbonitrile inhibited all of the tested mycobacterial strains with MIC within the range 12.5–25 μg/mL. Although not the most active, 4-NH₂ substituted compounds possessed the lowest in vitro cytotoxicity (hepatotoxicity), leading to selectivity index SI = 5.5 and SI >21.     

Synthesis and antimicrobial activity of novel amphiphilic aromatic amino alcohols

We report in this work the preparation and in vitro antimicrobial evaluation of novel amphiphilic aromatic amino alcohols synthesized by reductive amination of 4-alkyloxybenzaldehyde with 2-amino-2-hydroxymethyl-propane-1,3-diol. The antibacterial activity was determined against four standard strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and 21 clinical isolates of methicillin-resistant Staphylococcus aureus. The antifungal activity was evaluated against four yeast (Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis). The results obtained showed a strong positive correlation between the lipophilicity and the antibiotic activity of the tested compounds. The best activities were obtained against the Gram-positive bacteria (MIC = 2–16 μg ml^?1) for the five compounds bearing longer alkyl chains (4c–g; 8–14 carbons), which were also the most active against Candida (MIC = 2–64 μg ml^?1). Compound 4e exhibited the highest levels of inhibitory activity (MIC = 2–16 μg ml^?1) against clinical isolates of MRSA. A concentration of twice the MIC resulted in bactericidal activity of 4d against 19 of the 21 clinical isolates.     

Homocysteine and inflammation as main determinants of oxidative stress in the elderly

Oxidative stress is commonly observed in the elderly and could be involved in age-related diseases. However, the determinants of superoxide anion overproduction are not clearly understood. Superoxide anion production was evaluated using a lucigenin-based chemiluminescence method in 478 elderly subjects (304 women, 174 men; 79.5 ± 7.1 years). Homocysteine (HCy) metabolism (homocysteinemia, vitamin B12, plasma, and erythrocyte folates), inflammation (CRP, fibrinogen, α-1 acid glycoprotein), lipid parameters (total cholesterol, triglycerides, HDL and LDL cholesterol), and nutritional parameters (albumin, transthyretin) were determined. The results show that HCy levels (p < 0.001) and superoxide anion production (p = 0.04) increase with aging, but CRP does not. Highest HCy (> 20 μM) (OR 1.83 (1.09–3.07), p = 0.02) and CRP over 5 mg/L (adjusted OR 2.01 (1.15–3.51), p = 0.01) are the main determinants in superoxide anion production in the elderly. These clinical data are confirmed in an in vitro study using THP-1 monocyte-like cells. Incubation with HCy thiolactone (HTL) (0–200 μM) and LPS (0–20 ng/ml) dramatically enhances NADPH oxidase expression and activation. Moreover, a synergic action was evidenced for low concentrations of HTL (20 μM) and LPS (5 ng). Taken together, the clinical data and in vitro experiments support the hypothesis that moderate homocysteinemia and low-grade inflammation synergically enhance NADPH oxidase activity in the elderly.     

Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato,Ipomoea batatas (L.) Lam.

Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L.) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (2OGD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K_m = ∼10 μM, k_cat = ∼2.7 s⁻¹). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K_m = 7.3–14.0 μM, k_cat = 0.28–0.55 s⁻¹) and also of p-coumaroyl-CoA (K_m = 6.1–15.2 μM, k_cat = 0.28–0.64 s⁻¹) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly.     

Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry

A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC–MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 μl sample volume of biological matrixes can be extracted at 4 °C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC–MS/MS system without further clean-up and assayed across a linear range of 1–4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm × 100 mm, 3 μm) column and eluted at 200 μl/min with a tertiary mobile phase consisting of 20 mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 μl to 1 μl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 °C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1 m/z; 127.9 m/z), Erlotinib (393.9 m/z; 278.2 m/z), Sunitinib (399.1 m/z; 283.1 m/z) and Sorafenib (465.0 m/z; 251.9 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2 ± 3.8%, 11.2 nM; Erlotinib: 101.6 ± 3.7%, 12.7 nM; Sunitinib: 100.8 ± 4.3%, 12.6 nM; Sorafenib: 93.9 ± 3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.     

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.