Specific Activity Levels Research Articles

Tracking of Leisure Time Physical Activity during 28 yr in Adults

Physical activity plays an important role in the prevention of many serious diseases. To develop targeted strategies that encourage physical activity, knowledge of stability of physical activity levels over time is essential. The aim of this study was to examine tracking of leisure time physical activity in adults in Northern Norway during three decades. We followed 5432 women and men who attended the Tromsø Study in 1979-1980, as well as repeated examinations after 7 and 28 yr. Baseline age was 20-54 yr (mean age=35.8 yr). Physical activity was assessed by self-administered questionnaires. Tracking of physical activity, defined as maintenance of relative rank of physical activity level, was estimated by Spearman correlation coefficient and by weighted κ statistics. Tracking in terms of predictability of later values from earlier measurements was analyzed by generalized estimating equations. A higher-than-expected proportion of subjects maintained their physical activity level from examination 1 to 2 (58%) and 3 (53%). κ statistics showed agreement of 0.41 and 0.29, respectively. Belonging to a specific physical activity level at baseline increased the odds of belonging to the same category at later examinations (sedentary odds ratio (OR)=3.9 (95% confidence interval (CI)=3.5-4.4), moderately active OR=2.2 (95% CI=2.0-2.4), active OR=2.9 (95% CI=2.6-3.3), and highly active OR=14.0 (95% CI=8.7-22.5)). Being physically active in young adulthood increased the odds of being physically active later in life (moderately active OR=3.4 (95% CI=3.0-3.9), active OR=5.4 (95% CI=4.6-6.4), and highly active OR=13.0 (95% CI=7.4-22.8)). This study showed tracking of leisure time physical activity during 28 yr in a cohort of adults.

Detection, Isolation, Purification, and Characterization of Mannose Binding Lectin (ManBL) from Patients with Different Kidney Diseases and Healthy Individuals

The present study was designed to investigate the presence of mannose binding lectin (ManBL) in the sera and tissue samples of patients with tumoral (benign & malignant) kidney in addition to patients with non tumoral kidney affection, in order to establish the possibility of using serum and tissue ManBL test for diagnosis and epidemiological testing of kidney cancers. Participants of the present study were 96 patients at the age range 12-80 years; they were attending by different hospitals in Najaf (basically) and Karbala, and 46 healthy individuals within the same age range of the studying patients groups. Specific hemagglutination activity levels were revealed a significant increased (p < 0.001) in the sera and tissues of the patients with the malignant tumors specimens in comparison to that of pathological controls and healthy individuals. Serum and tissue hemagglutination activity levels were significantly higher (p < 0.001) in patients with metastatic disease compared with patients with localized tumors. While, the levels of the hemagglutination activity were approximate at patients with the end malignant kidney tumors stages. Upon electrophoresis of the study samples, the results reflected presence of changes in some proteins and glycoproteins bands presents in these samples. Using hydrophobic affinity chromatography, ManBLs were purified from sera of malignant, benign kidney tumors and non tumoral kidney diseases in addition to healthy individuals groups. The purified lectins were found to be glycoprotein with 77.45 KD as an approximate molecular weight and its sugar content was equal to13.5%. The maximum concentration of purified ManBL was found at patients with malignant kidney tumors.The results revealed that the highest hemagglutination activity of purified ManBL occurred with O + RBCs, at pH 7.4, and 37˚C. ManBL (regardless of its origin) lost the hemagglutination activity, completely, in the presence of EDTA. A result that indicates that the present purified the human ManBLs is a calcium dependent type.

Erratum to: Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium Pseudomonas frederiksbergensis

A gram-negative psychrophilic bacterium, with potential for biodegradation of long-chain n-alkanes was isolated from ice samples collected in Spitzbergen, Denmark. On the basis of results of biochemical and morphological tests and sequence analysis of 16S rRNA, the strain was identified as Pseudomonas frederiksbergensis. In this work, a short-chain NAD+-dependent alcohol dehydrogenase (alcDH) (Accession number: AAR13804) from the P. frederiksbergensis was cloned and transformed in E. coli BL21 (3DE) competent cells. The alcDH activity was highest in the crude extract of cells induced with 1.0 mM IPTG. The recombinant alcDH enzyme was purified to 93.4% homogeneity using three consecutive purification steps including ammonium sulphate, Q-Sepharose Fast Flow column and gel filtration chromatography employing Superdex 200 10/30 HR column. Enzyme enrichment and yield levels of 31.4 folds and 25.5%, respectively, were achieved. While the subunit molecular mass of the enzyme was determined on SDS-PAGE to be ~38 kDa, the aggregated native form of the enzyme had a molecular mass of ~238 kDa by gel filtration analysis. Reaction conditions optima for the recombinant alcDH were determined with propan-1-ol as the substrate. While the optimum pH was 9, the optimum temperature was 35 °C. The alcDH enzyme exhibited moderate thermal stability with half-lives of 150 min at 55 °C, 27 min at 65 °C and 8 min at 75 °C. Results for kinetic parameters indicated that the apparent K m value for alcDH with propan-1-ol as the substrate was found to be 1.42 mM and the V max value was 0.63 mmol mg−1 min−1. Experimental evidence revealed that the recombinant alcDH exhibited a wide range of substrate specificity, with higher levels of specific activity for aliphatic alcohols as compared to secondary alcohols. Taken together, the present study highlights the potential of alcDH as a member of cold-adapted enzymes in several key biotechnological applications including environmental bioremediation and biotransformations. It is envisaged that, with the ongoing screening of microorganisms and metagenomes, directed evolution approaches and the subsequent overexpression of recombinant proteins, more enzymes will be found that are suitable for bioremediation purposes.

WASTE CLASSIFICATION OF 17×17 KOFA SPENT FUEL ASSEMBLY HARDWARE

Metal waste generated from the pyroprocessing of 10 MtU of spent fuel was classified by comparing the specific activity of a relevant radionuclide with the limit value of the specific activity specified in the Korean acceptance criteria for a lowand intermediate-level waste repository. A Korean Optimized Fuel Assembly design with a 17<TEX>${\times}$</TEX>17 array, an initial enrichment of 4.5 weight-percent, discharge burn-up of 55 GWD/MtU, and a 10-year cooling time was considered. Initially, the mass and volume of each structural component of the assembly were calculated in detail, and a source term analysis was subsequently performed using ORIGEN-S for these components. An activation cross-section library generated by the KENO-VI/ORIGEN-S module was utilized for top-end and bottom-end pieces. As a result, an Inconel grid plate, a SUS plenum spring, a SUS guide tube subpart, SUS top-end and bottom-end pieces, and an Inconel top-end leaf spring were determined to be unacceptable for the Gyeongju low- and intermediate-level waste repository, as these waste products exceeded the acceptance criteria. In contrast, a Zircaloy grid plate and guide tube can be placed in the Gyeongju repository. Non-contaminated Zircaloy cladding occupying 76% of the metal waste was found to have a lower level of specific activity than the limit value. However, Zircaloy cladding contaminated by fission products and actinides during the decladding process of pyroprocessing was revealed to have 52 and 2 times higher specific activity levels than the limit values for alpha and <TEX>$^{90}Sr$</TEX>, respectively. Finally, it was found that 88.7% of the metal waste from the 17<TEX>${\times}$</TEX>17 Korean Optimized Fuel Assembly design should be disposed of in a deep geological repository. Therefore, it can be summarized that separation technology with a higher decontamination factor for transuranics and strontium should be developed for the efficient management of metal waste resulting from pyroprocessing.

Influence of Osmotic Stress on Fermentative Production of Succinic Acid by Actinobacillus succinogenes

This study investigated the influence of osmotic stress on succinic acid production by Actinobacillus succinogenes NJ113. Both cell growth and succinic acid production were inhibited with the increase in osmotic stress of the medium. The use of three different osmoprotectants in the production of succinic acid was studied in order to decrease the inhibitory effects of osmotic stress during fermentation. Results indicated that proline offers optimal osmoprotection in the production of succinic acid by A. succinogenes NJ113. In tests of batch fermentation, the maximum cell concentration was observed to be 5.36g DCW/L after the addition of 25mmol/L proline to the fermentation medium. The cell concentration was 24% higher than that noted for the control. A total quantity of 56.2g/L of succinic acid was produced, with a production rate of 1g/L per hour, after 56h of fermentation. The concentration and productivity of succinic acid was observed to be increased by 22.2% and 22%, respectively, as compared with the control. The specific activity levels of key enzymes in the metabolic network was noted to be higher following the addition of proline, particularly in the later stages of fermentation. This method of enhancing succinic acid production by the addition of an osmoprotectant may potentially provide an alternative approach for enhanced production of other organic acids.

The role of CTSH in narcolepsy susceptibility and autoimmunity (47.16)

Abstract Narcolepsy, a disease associated with HLA-DQB1*0602, is caused by the selective autoimmune destruction of 70,000 hypocretin neurons in the brain. Recently we identified Cathepsin-H as a novel Narcolepsy susceptibility locus. SNP rs3825932 was associated with Narcolepsy in our set of 807 Caucasian cases vs. 1074 controls (p= 0.002 OR 1.24). The same SNP marker, was previously reported to be associated Type 1 Diabetes in two studies. Cathepsin H is a member of the papain-like family, and participates in intracellular protein degradation. CTSH have also been suggested to be involved in apoptosis and neurodegeneration. CTSH expression levels in peripheral white blood cells are strongly correlated with CTSH genotype at rs1036938 (p&amp;lt;0.0001). Presence of the disease associated C allele is correlated with a lower expression of the CTSH, in a dose dependent manner, in CD8 (T lymphocytes, NK) and CD4 (TH, regulatory, monocytes, macrophages, dendritic) cells, but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with lower CTSH specific activity levels (p=0.029). The genetic association, and results from expression and activity studies strongly suggest a role for CTSH in the development of Narcolepsy. CTSH is implicated in at least two highly selective autoimmune diseases, therefore understanding the role of CTSH in Narcolepsy susceptibility may yield important insights into the development of that and other autoimmune diseases.

Digestive Enzymes in Larvae and Juveniles of Farmed Sharpsnout Seabream (Diplodus puntazzo) (Cetti, 1777)

The ontogenetic development and pattern of main digestive enzyme activities (proteases, carbohydrases and lipases) were investigated in farmed sharpsnout seabream Diplodus puntazzo larvae and juveniles, during the passage from the larval stage to adult (from 21 to 277 days after hatching - DAH). Larvae showed exponential growth, thus confirming that Rotifers and Artemia nauplii are suitable preys for early feeding of D. puntazzo larvae. Pepsin activity was low in the larvae, but it increased afterwards in relation with stomach development. In juveniles, levels of specific activity of trypsin, chymotrypsin and elastase were lower compared to that recorded in larvae. A decrease in the activity of the alkaline proteases could be related to an increase in the functionality of stomach. Qualitative and quantitative assay of glycosidases (� -amylase, cellulase and maltase) showed high glycosidase activity, indicating that D. puntazzo is capable of digesting carbohydrates in its diet. Levels of lipase activity in juveniles were higher than those found in larvae. Our results didn't show an inductive character of lipases, since in juveniles the activity levels were almost constant even if the fat content of compound diet varied (from 7.0 % to 20 %). The rapid changes in the digestive activities of D. puntazzo larvae supported the existence of well established induction and regulation digestive mechanisms. Present results also confirm that D. puntazzo larvae have complete digestive enzymatic equipment that increased during larval ontogeny and enable larvae to digest and absorb nutrients contained in live preys once exogenous feeding occurred. The enzymatic pattern is correlated with omnivorous feeding habits of the species. Moreover, the patterns of activity of the main digestive enzymes of D. puntazzo larvae and juveniles indicated the early functional development of their digestive system and suggested this species as a good candidate for Mediterranean aquaculture using the compound diet.

Enhanced functional stability of plasminogen activator inhibitor-1 in patients with livedoid vasculopathy

Livedoid vasculopathy (LV) is a chronic, recurrent, painful cutaneous disease with distinctive clinical features and an uncertain etiology. The skin lesions are recognizable by focal purpura, depigmentation and shallow ulcers. Thrombophilic conditions occur frequently in patients with LV. While no definitive treatment exists for LV, smoking cessation, antiplatelet therapy, immunosuppressive treatment, and anabolic steroids are often included in the therapeutic ladder. Recently, a possible link between LV and impaired fibrinolysis was established as cutaneous LV lesions responded to tissue plasminogen activator (t-PA) infusion suggesting that inhibition of the fibrinolysis through plasminogen activator inhibitor-1 (PAI-1) activity may determine the disease course in patients with LV. In this study, we investigated PAI-1 antigen (Ag) and activity levels in 20 patients with biopsy proven LV (mean age 26 ± 11, M/F = 7/13, median disease duration 3.5 years). All patients received antiplatelet treatment with aspirin and/or dipyrimadole and 14 patients received anabolic steroids or immunosuppressive treatment. Fasting PAI-1 Ag and activity levels were measured at 9 AM in all patients. Both Ag (34 (26) ng/ml) (median (interquartile range)) and specific activity (17 (23) IU/fmole) levels of PAI-1 were moderately elevated in LV patients compared to the controls, however, PAI-1 kinetic studies demonstrated markedly enhanced stability of PAI-1 activity in plasma from patients with LV. Specific activity at 16 h was significantly higher than expected specific activity levels (7 (11) vs. 0.07 (0.09) IU/fmole, P < 0.01). While the exact mechanism of increased stability of PAI-1 activity is not known, it may be due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of LV, and systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative in patients with LV.

Expression of manganese superoxide dismutase in rat blood, heart and brain during induced systemic hypoxia

Background: Hypoxia results in an increased generation of ROS. Until now, little is known about the role of MnSOD - a major endogenous antioxidant enzyme - on the cell adaptation response against hypoxia. The aim of this study was to determine the MnSOD mRNA expression and levels of specific activity in blood, heart and brain of rats during induced systemic hypoxia. Methods: Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia in an hypoxic chamber (at 8-10% O2) for 0, 1, 7, 14 and 21 days, respectively. The mRNA relative expression of MnSOD was analyzed using Real Time RT-PCR. MnSOD specific activity was determined using xanthine oxidase inhibition assay. Results: The MnSOD mRNA relative expression in rat blood and heart was decreased during early induced systemic hypoxia (day 1) and increased as hypoxia continued, whereas the mRNA expression in brain was increased since day 1 and reached its maximum level at day 7. The result of MnSOD specific activity during early systemic hypoxia was similar to the mRNA expression. Under very late hypoxic condition (day 21), MnSOD specific activity in blood, heart and brain was significantly decreased. We demonstrate a positive correlation between MnSOD mRNA expression and specific activity in these 3 tissues during day 0-14 of induced systemic hypoxia. Furthermore, mRNA expression and specific activity levels in heart strongly correlate with those in blood.

福島第一原発事故に起因する放射性物質による生活環境における土壌汚染に関する調査

Radioactive materials were released to the general environment due to the accident at the Fukushima Daiichi nuclear power station. The released radioactive materials fell and contaminated the land mainly in the Tohoku and Kanto areas of Japan. We surveyed the air dose rate in relation to the pave condition of the land, and investigated the contamination level in some nonpaved areas at the center of Fukushima City, Koriyama City, and Nasushiobara City. From the survey results, the dose rates of the nonpaved areas were found to be higher than those of the paved areas, and the dose rates of the paved areas depend on the paving materials of the area. The contamination level of the nonpaved area in Nasushiobara City was below the regulation level of specific activities in a radiation-controlled area in Japan. However, the contamination levels in the nonpaved areas in Fukushima City and Koriyama City were above the regulation level.

Study of Various Presentation Forms for a Peptide Mimetic of Neisseria meningitidis Serogroup B Capsular Polysaccharide

The formulation of a broadly protective vaccine to prevent the serogroup B Neisseria meningitidis (MenB) disease is still an unmet medical need. We have previously reported the induction of bactericidal and protective antibodies against MenB after immunization of mice with a phage-displayed peptide named 4 L-5. This peptide mimics a capsular polysaccharide (CPS) epitope in MenB. With the aim of developing vaccine formulations that could be used in humans, we evaluate in this study various forms of presentation to the immune system of the 4 L-5 sequence, based on synthetic peptides. We synthesized the following: (i) a linear 4 L-5 peptide, (ii) a multiple antigen peptide containing four copies of the 4 L-5 sequence (named MAP), which was then dimerized, and the product named dimeric MAP, and (iii) a second multiple antigen peptide, in this case with two copies of the 4 L-5 sequence and a copy of a T-helper cell epitope of tetanus toxoid, which was then dimerized and the product named MAP-TT. The linear peptide, the MAP, and the dimeric MAP were conjugated to the carrier protein P64K by different conjugation methods. Plain antigens and antigens coupled to P64K were used to immunize BALB/c mice. Of those variants that gave immunogenic results, MAP-TT rendered the highest levels of specific antipeptide IgG antibodies and serum bactericidal activity. These results can find application in the development of meningococcal vaccine candidates and in peptide-based vaccines strategies.

Effects of dissolved oxygen availability and culture biomass at induction upon the intracellular expression of monoamine oxidase by recombinant E. coli in fed batch bioprocesses

The effects of oxygen availability and induction culture biomass upon production of an industrially important monoamine oxidase (MAO) were investigated in fed-batch cultures of a recombinant E. coli. For each induction cell biomass 2 different oxygenation methods were used, aeration and oxygen enriched air. Induction at higher biomass levels increased the culture demand for oxygen, leading to fermentative metabolism and accumulation of high levels of acetate in the aerated cultures. Paradoxically, despite an almost eight fold increase in acetate accumulation to levels widely reported to be highly detrimental to protein production, when induction wet cell weight (WCW) rose from 100% to 137.5%, MAO specific activity in these aerated processes showed a 3 fold increase. By contrast, for oxygenated cultures induced at WCW's 100% and 137.5% specific activity levels were broadly similar, but fell rapidly after the maxima were reached. Induction at high biomass levels (WCW 175%) led to very low levels of specific MAO activity relative to induction at lower WCW's in both aerated and oxygenated cultures. Oxygen enrichment of these cultures was a useful strategy for boosting specific growth rates, but did not have positive effects upon specific enzyme activity. Based upon our findings, consideration of the amino acid composition of MAO and previous studies on related enzymes, we propose that this effect is due to oxidative damage to the MAO enzyme itself during these highly aerobic processes. Thus, the optimal process for MAO production is aerated, not oxygenated, and induced at moderate cell density, and clearly represents a compromise between oxygen supply effects on specific growth rate/induction cell density, acetate accumulation, and high specific MAO activity. This work shows that the negative effects of oxygen previously reported in free enzyme preparations, are not limited to these acellular environments but are also discernible in the sheltered environment of the cytosol of E. coli cells.

Characterization of Antibodies Induced by Vaccination with Hepatitis C Virus Envelope Glycoproteins

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 were used with MF59 adjuvant as a candidate vaccine for a phase 1 safety and immunogenicity trial. Ten of 41 vaccinee serum samples displayed a neutralization titer of ⩾1:20 against vesicular stomatitis virus (VSV)-HCV pseudotype, 15 of 36 serum samples tested had a neutralization titer of ⩾1:400 against human immunodeficiency virus (HIV)-HCV pseudotype, and 10 of 36 serum samples tested had a neutralization titer of ⩾1:20 against cell culture-grown HCV genotype 1a. Neutralizing serum samples had increased affinity levels and displayed >2-fold higher specific activity levels to well-characterized epitopes on E1/E2, especially to the hypervariable region 1 of E2.

Temporal and functional pattern of secreted enzyme activities in an ectomycorrhizal community

The ectomycorrhizal community of an oak forest has been monitored monthly throughout fifteen months. Eight enzymatic activities secreted by the ectomycorrhizal root tips and involved in the mobilization of nutrients from soil organic matter have been measured using microplate assays, resulting in potential activity patterns of individual fungal species. Both the species structure of the community and the specific activity level of each individual species changed with the season and soil horizon. This versatility may be an adaptative response of the ectomycorrhizal fungal community to a highly variable environment. The results also suggest that some ectomycorrhizal fungi behave as occasional saprobes and contribute to the decomposition of soil organic matter and nutrient cycling together with true saprotrophic fungi.

18F-Labeling and evaluation of novel MDL 100907 derivatives as potential 5-HT2A antagonists for molecular imaging

The serotonergic system, especially the 5-HT2A receptor, is involved in various diseases and conditions. It is a very interesting target for medicinal applications. Two novel 5-HT2A tracers, namely, [(18)F]DD-1 and the enantiomeric pure (R)-[(18)F]MH.MZ, were radiolabeled by (18)F-fluoroalkylation of the corresponding desmethyl analogue. In vitro binding autoradiography on rat brain slices was performed to test the affinity and selectivity of these tracers. Moreover, first microPET experiments of (R)-[(18)F]MH.MZ were carried out in Sprague-Dawley rats. [(18)F]DD-1 (K(i)=3.23 nM) and (R)-[(18)F]MH.MZ (K(i)=0.72 nM) were (18)F-fluoroalkylated by the secondary synthon [(18)F]FETos in a radiochemical yield (RCY) of >70%. The final formulation for both tracers took no longer than 100 min with an overall RCY of approximately 40%. It provided [(18)F]tracers with a purity >96% and a typical specific activity of 25-35 GBq/mumol. Autoradiographic images of (R)-[(18)F]MH.MZ (5) and [(18)F]DD-1 (4) showed excellent visualization and selectivity of the 5-HT2A receptor for (R)-[(18)F]MH.MZ and less specific binding for [(18)F]DD-1. The binding potential (BP) of (R)-[(18)F]MH.MZ was determined to be 2.6 in the frontal cortex and 2.2 in the cortex (n=4), whereas the cortex-to-cerebellum ratio was determined to be 3.2 at steady state (n=4). Cortex-to-cerebellum ratios of (R)-[(18)F]MH.MZ were almost twice as much as compared with the racemic [(18)F]MH.MZ. Thereby, equal levels of specific activities were used. High uptake could be demonstrated in cortex regions. Labeling of both novel tracers was carried out in high RCY. Autoradiography revealed (R)-[(18)F]MH.MZ as a very selective and affine 5-HT2A tracer (K(i)=0.72 nM), whereas [(18)F]DD-1 showed no reasonable distribution pattern on autoradiographic sections. Moreover, results from microPET scans of (R)-[(18)F]MH.MZ hint on improved molecular imaging characteristics compared with those of [(18)F]MH.MZ. Therefore, (R)-[(18)F]MH.MZ appears to be a highly potent and selective serotonergic PET ligand in small animals.

Computational prediction and experimental validation of 5 DNA‐binding residues of human flap endonuclease 1 (hFEN1)

Human FEN1 is a structure‐specific DNA nuclease involved in removing 5′ RNA primers of Okazaki fragments and in long‐patch base excision repair. Defects in FEN1 lead to increased genomic instability. Identification of DNA‐binding residues will further elucidate the molecular mechanisms behind genome instability initiation and facilitate cancer therapy linked to hFEN1 mutations. No crystal structure data is available of hFEN1 actively bound to DNA; however, a structure of an hFEN1 homolog, T4 phage RNase H in its active form (pdb 2ihn), is available as well as hFEN1 complexed with PCNA in the inactive form (pdb 1ul1). The 2ihn pdb structure shows 31 DNA‐interacting residues. Computational structural alignment of the two X‐ray crystal structures has revealed 5 residues within the 31 subset of RNase H that have a seemingly structurally conserved equivalent in hFEN1; these are K74, L29, V30, F53, and H174. These conserved equivalents in hFEN1 may therefore interact with DNA. To validate our predictions, we use HPLC to measure specific activity levels of hFEN1 mutant proteins versus the wildtype using fluorescein‐labeled DNA substrate. We found that the specific activities of the mutants are impaired relative to the wildtype. We will measure DNA binding activity of hFEN1 mutants using gel shift with biotin‐labeled DNA substrate to validate if the results we observe are due to defects in DNA substrate binding.This work is funded by NIH R01CA076734: Fen1 in genome stability and cancer.

Cryopreservation of Autologous Fat Grafts Harvested With the Coleman Technique

The viability of fat grafts harvested with an established technique after cryopreservation remains unknown. This study was conducted in vitro to evaluate the viability of autologous fat grafts harvested with the Coleman technique and subsequently preserved with our preferred cryopreservation method. Eight adult females were enrolled in this study. In each patient, 10 mL of fat grafts were harvested with the Coleman technique by a single surgeon from the lower abdomen. In group 1, 5 mL of fresh fat grafts were mixed with cryoprotective agents and underwent cryopreservation with controlled slow cooling and fast rewarming. In group 2, 5 mL of fresh fat grafts without cryopreservation from the same patient served as a control. The fat graft samples from both groups were evaluated with trypan blue vital staining, glycerol-3-phophatase dehydrogenase assay, and routine histology. Viable adipocyte counts were found similar in both group 1 and group 2 (3.46 +/- 0.91 vs. 4.12 +/- 1.11 x 10/mL, P = 0.22). However, glycerol-3-phophatase dehydrogenase activity was significantly lower in group 1 compared with group 2 (0.47 +/- 0.09 vs. 0.66 +/- 0.09 u/mL, P < 0.001). Histologically, the normal structure of fragmented fatty tissues was found primarily in both groups. Our results indicate that autologous fat grafts harvested with the Coleman technique and preserved with our preferred cryopreservation method have a normal histology with near the same number of viable adipocytes as compared with the fresh fat grafts. However, those cryopreserved fat grafts appear to have a less optimal level of adipocyte specific enzyme activity compared with the fresh ones and thus may not survive well after they are transplanted without being optimized.

Differential in vitro responses of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea. 2. Aromatic biosynthesis

Phenylalanine ammonia lyase (PAL)-specific activity levels were measured in suspension-cultured cells of six rice cultivars following treatment with cell wall hydrolysates prepared from seven Pyricularia grisea strains. Early after elicitation, even low hydrolysate concentrations were able to induce a significant increase in enzyme levels. However, neither rice genotypes showing differential sensitivity to blast reacted differently, nor did elicitors obtained from various pathotypes induce different reactions. At a later stage, higher hydrolysate concentrations were required to trigger maximal enzyme induction. Also in this case, only slight variations were detected in suspension cultures of a given cultivar treated with different elicitors. On the contrary, highly significant differences were observed among plant genotypes. A clear relationship was evident between the mean increase in PAL activity and the overall resistance to blast at the plant level. This trait could therefore represent a useful tool in selection for increased blast tolerance.

Cosubstrate effect on xylose reductase and xylitol dehydrogenase activity levels, and its consequence on xylitol production by Candida tropicalis

Due to its anticariogenic properties, the five-carbon sugar alcohol xylitol is finding increasing use in food industry as a functional sweetener. Although xylitol is currently manufactured by the chemical reduction of xylose, its biological production would be attractive because of lower costs and better organoleptic features. In xylose-fermenting yeasts, the substrate is directly converted to xylitol by an NAD(P)H-dependent reductase (XR). Xylitol is oxidized in turn by an NAD +-dependent dehydrogenase (XDH) yielding xylulose, which is channelled into the pentose phosphate pathway. In the absence of other carbon sources, a significant fraction of xylitol is used to fuel cell growth. To avoid excess yield loss and maximize xylitol production, a series of mono- and disaccharides were tested as cosubstrates for a hyper-acidophilic strain of Candida tropicalis. Their effect upon XR and XDH specific activity levels was evaluated, and the resulting expression of the inducible pathway and the XR:XDH ratio were related to either non-optimized or optimized yields. Glucose strongly inhibited xylose reduction, whereas galactose stimulated xylitol utilization. On the contrary, the addition of low concentrations of maltose significantly increased biomass growth and xylitol accumulation.

A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OL T

Caldicellulosiruptor owensensis strain OL T (DSM 13100) is an obligately anaerobic, extreme thermophilic bacterium that is capable of utilizing a broad range of carbohydrates and producing H 2 as a metabolic by-product. The performance of C. owensensis on glucose and xylose was analyzed in lab-scale bioreactors to assess its potential use in biohydrogen production. Acetate, H 2, and CO 2 were the main end products during exponential growth of the organism on either sugar. Lactate production was triggered during the transition into the stationary phase and was associated with an increase in the levels of specific l-lactate dehydrogenase activity. In addition, minor amounts of ethanol and propionate could be detected. H 2 and acetate yields were lower on xylose than on glucose, marking an opposite trend to biomass and lactate yields. The influence of elevated H 2 partial pressure on product distribution was more dramatic in xylose-fermenting cultures. Replacement of yeast extract in the medium with a standard vitamins solution improved H 2 yield on both sugars, where it reached 100% of the theoretical maximum, i.e. 4 mol per mol hexose, on glucose. By using the defined medium, both the maximum specific growth rate and the maximum volumetric H 2 production rate of C. owensensis increased significantly on glucose and almost doubled on xylose. Screening other sugars besides glucose and xylose revealed a clear sugar-dependent product-distribution pattern and a direct correlation between biomass and lactate yields, which might be explained considering energy metabolism of the cells. The organism is proposed as a new candidate for biohydrogen production at high yields.