Characterisation of a capsular polysaccharide from Moraxella nonliquefaciens CCUG 348T.

Moraxella nonliquefaciens is a commensal of the human upper respiratory tract (URT) but on rare occasions is recovered in cases of ocular, septic and pulmonary infections. Hence there is interest in the pathogenic determinants of M. nonliquefaciens, of which outer membrane (OM) structures such as fimbriae and two capsular polysaccharide (CPS) structures, →3)-ß-D-GalpNAc-(1→5)-ß-Kdop-(2→ and →8)-α-NeuAc-(2→, have been reported in the literature. To further characterise its surface virulence factors, we isolated a novel CPS from M. nonliquefaciens type strain CCUG 348T. This structure was elucidated using NMR data obtained from CPS samples that were subjected to various degrees of mild acid hydrolysis. Together with GLC-MS data, the structure was resolved as a linear polymer composed of two GalfNAc residues consecutively added to Kdo, →3)-ß-D-GalfNAc-(1→3)-α-D-GalfNAc-(1→5)-α-(8-OAc)Kdop-(2→. Supporting evidence for this material being CPS was drawn from the proposed CPS biosynthetic locus which encoded a potential GalfNAc transferase, a UDP-GalpNAc mutase for UDP-GalfNAc production and a putative CPS polymerase with predicted GalfNAc and Kdo transferase domains. This study describes a unique CPS composition reported in Moraxella spp. and offers genetic insights into the synthesis and expression of GalfNAc residues, which are rare in bacterial OM glycans.

Disruption of p-coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure.

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

Nanocidal Effect of Rice Husk-Based Silver Nanoparticles on Antioxidant Enzymes of Aphid.

Green synthesis of nanoparticles using plant-based extracts is momentously used in different fields of science because of their environment-friendly nature and cost-effectiveness. In the present study, silver nanoparticles were synthesized by using rice husk (non-toxic agricultural by-product) to determine their efficacy against aphid's (Sitobion avanae) mortality and antioxidant enzymes. UV-VIS spectroscopy of synthesized nanoparticles showed the maximum absorption peak at 440 nm, FTIR exhibited different peaks, and SEM confirmed the roughly spherical shape and 70-80 nm size of silver nanoparticles. Aphids were reared on wheat seedlings in the laboratory at 20-25 °C and 16:8 (light:dark) photoperiod. Insecticidal bioassays were conducted on aphids at three different concentrations (200 ppm, 400 ppm, 600 ppm) of nanoparticles for 2 days. Results showed the highest mortality of aphids being 93.3% at 600 ppm nanoparticle concentration after 2 days while the lowest mortality was observed at 200 ppm. Furthermore, the effect of silver nanoparticles on antioxidant enzymes was studied. Results of enzyme assays revealed that enzyme activities of catalase and glutathione-s-transferase increased in response to increased nanoparticle concentration. The current findings suggested that silver nanoparticles have probation for replacing commercially available insecticides for combating pests.

Functional analysis and enzyme characterization of mannose-1-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis cell wall consist variety of mannose containing glycoconjugates including lipomannan (LM) and lipoarabinomannan (LAM). These lipoglycans are involved in cell wall integrity and play role in virulence of M. tuberculosis by modulating host immune response. GDP-mannose, required for the synthesis of lipoglycans, is catalyzed by enzyme Mannose-1-phosphate guanylyl transferase (ManB). The enzyme with similar function has been studied in variety of species of prokaryotes and eukaryotes. However, biological role of ManB and its enzymatic activity remains uncharacterized in M. tuberculosis. In present study, we elucidated the role of enzyme by constructing manB knockdown strain of M. tuberculosis H37Ra. The manB knockdown decreased the cell growth and also effected the morphology of M. tuberculosis by altering the permeability of cell membrane. These findings provide the understanding on ManB function and suggesting that ManB could be the potential target for novel anti-tuberculosis drug. Furthermore, we also characterized ManB enzyme by establishing 96 well plate colorimetric assay and determined the kinetic properties including initial velocity, optimum temperature, optimum pH and other kinetic parameters. Our established assay will be helpful for further high throughput screening of potential inhibitors against ManB.

Allergen Profile of London Plane Tree Pollen: Clinical and Molecular Pattern in Central Spain.

BACKGROUND AND OBJECTIVES: Platanus acerifolia (London plane tree) is a deciduous tree of the Platanaceae family. Sensitization to this plant varies with geography. Madrid, located in central Spain, has one of the highest London plane tree pollen concentration levels on the Iberian Peninsula. We evaluated both the clinical characteristics and the molecular sensitization pattern of patients with allergy to London plane tree pollen in the region of Madrid. METHODS: Thirty-eight patients allergic to London plane tree pollen were selected according to their clinical symptoms and positive results in skin prick testing and/or specific IgE determination. Serum was collected, and allergen components were evaluated using immunodetection techniques as well as ImmunoCAP. The IgE-binding proteins detected were identified and characterized using mass spectrometry. RESULTS: Analysis of serum samples from allergic patients revealed 9 IgE-binding bands in London plane tree pollen extract. Among these, the 45-kDa protein, which corresponded to Pla a 2, was detected in 76.3% of patients. However, the 18-kDa (Pla a 1) and 9-kDa (Pla a 3) bands were detected in 44.7% and 23.7% of sera, respectively. These results were confirmed using purified proteins. Characterization of the allergen revealed the 27-kDa protein to be glutathione-S-transferase. CONCLUSIONS: The molecular profile of patients sensitized to London plane tree pollen differs from that reported in studies from other locations. In the population we studied, the prevalence of Pla a 2 was higher than that of Pla a 1 and Pla a 3. In addition, the minor allergen previously referred to as Pla a 4 was characterized as glutathione-S-transferase.

Gut microbiota composition reflects disease severity and dysfunctional immune responses in patients with COVID-19.

OBJECTIVE: Although COVID-19 is primarily a respiratory illness, there is mounting evidence suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if any, resolve with clearance of the SARS-CoV-2 virus. METHODS: In this two-hospital cohort study, we obtained blood, stool and patient records from 100 patients with laboratory-confirmed SARS-CoV-2 infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance of SARS-CoV-2. Gut microbiome compositions were characterised by shotgun sequencing total DNA extracted from stools. Concentrations of inflammatory cytokines and blood markers were measured from plasma. RESULTS: Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals irrespective of whether patients had received medication (p<0.01). Several gut commensals with known immunomodulatory potential such as Faecalibacterium prausnitzii, Eubacterium rectale and bifidobacteria were underrepresented in patients and remained low in samples collected up to 30 days after disease resolution. Moreover, this perturbed composition exhibited stratification with disease severity concordant with elevated concentrations of inflammatory cytokines and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase and gamma-glutamyl transferase. CONCLUSION: Associations between gut microbiota composition, levels of cytokines and inflammatory markers in patients with COVID-19 suggest that the gut microbiome is involved in the magnitude of COVID-19 severity possibly via modulating host immune responses. Furthermore, the gut microbiota dysbiosis after disease resolution could contribute to persistent symptoms, highlighting a need to understand how gut microorganisms are involved in inflammation and COVID-19.

De novo assembly of the Mylia taylorii transcriptome and identification of sesquiterpene synthases.

Mylia taylorii is an ancient nonseed land plant that accumulates various sesquiterpenes with insecticidal and antibacterial activities. Recently, microbial-type sesquiterpene synthases (STSs) with atypical aspartate-rich metal ion binding motifs have been identified in some liverworts. Here, transcriptome analysis of M. taylorii was performed to identify M. taylorii sesquiterpene synthases (MtSTSs) that are potentially involved in sesquiterpene biosynthesis and diversity. A total of 255,669 unigenes were obtained with an average length of 963 bp in the transcriptome data of M. taylorii, among which 148,093 (57.92%) unigenes had BLAST results. Forty-eight unigenes were related to the sesquiterpene backbone biosynthesis according to KEGG annotation. In addition, MtSTS1, MtSTS2 and MtSTS3 identified from putative MtSTSs display sesquiterpene catalytic activities on the basis of functional characterizations in yeast. Interestingly, MtSTSs exhibit a noncanonical metal ion binding motif and the structural composition of a single α-domain, which are features of microbial STSs instead of archetypical plant STSs. This study revealed new microbial-type STS members of nonseed plants, and functionally identified that MtSTSs may contribute to the investigation of the biosynthesis and biological role of sesquiterpenes in M. taylorii.

Elevación de enzimas hepáticas inducida por COVID-19 en embarazada / Elevated liver enzimes induced by COVID-19 in pregnancy

INTRODUCCIÓN: Las alteraciones del perfil hepático durante el embarazo ocurren en 3-5% de las gestantes. Una nueva etiología que se ha presentado en el contexto de pandemia actual es el síndrome respiratorio agudo severo relacionado con el nuevo coronavirus (SARS-CoV-2). Éste es responsable de alteraciones hepáticas en 2 a 11% de la población general infectada por este virus, y de hasta un 30% en las embarazadas que se infectan con SARS-CoV-2. Con el objetivo de mostrar una presentación poco frecuente del SARS-CoV-2 se expone un caso clínico de elevación de transaminasas en embarazada inducida por este nuevo virus. CASO CLÍNICO: Paciente de 36 años, cursando embarazo de 20+6 semanas, consulta por dolor abdominal asociado a ictericia y coluria. Se solicita estudio donde destaca elevación de transaminasas. Ecografía abdominal con vía biliar fina. Se descartan diferentes etiologías de hepatitis aguda y crónica (dada la falta de antecedentes). Finalmente se solicita PCR para COVID-19 que resulta positiva. CONCLUSIÓN: Luego de un estudio exhaustivo de diferentes etiologías de elevación de transaminasas, se atribuye esta alteración enzimática a SARS-CoV-2. Se decide seguimiento ambulatorio estricto con pruebas hepáticas cada dos semanas. La paciente evoluciona estable con exámenes normales luego de un mes desde que se indica el alta hospitalaria. Después de descartar etiologías frecuentes de elevación de transaminasas durante el embarazo, sugerimos solicitar el estudio de este virus con PCR para COVID-19, ya que podría ser una presentación poco frecuente de SARS-CoV-2.

INTRODUCTION: Approximately 3-5% of women present alterations of hepatic enzymes during pregnancy. Under the new circumstances that the world is facing with the SARS-COV2 pandemic, a new etiology for hepatic enzyme alterations has risen. The severe acute respiratory syndrome that the novel coronavirus causes is responsible for hepatic enzyme alterations in 2 to 11% of the sick population that did not have a previous underlying hepatic condition. Furthermore, hepatic enzyme alterations in pregnant women infected with SARS-COV2 presents in up to 30% of the cases. An infrequent presentation of SARS-COV2 is presented as our clinical case. CLINICAL CASE: A 36-year-old patient with a 20+6 week pregnancy presents abdominal pain, jaundice and choluria. General blood workup shows elevated transaminases. The abdominal ultrasound revealed a thin bile duct. Acute and chronic hepatitis etiologies were discarded. Finally, a PCR of COVID-19 was solicited, which turned out to be positive. CONCLUSIÓN: After an exhaustive study to determine the etiology of the elevated transaminases, the hepatic alterations were attributed to SARS-COV2 infection. A conservative management was adopted, with outpatient follow-up with liver testing every two weeks. The patient progresses with a stable steady decline in hepatic enzyme levels, and one-month post hospital discharge, her transaminases had reached normal values. Based on this clinical case, after ruling out frequent etiologies for elevated transaminases during pregnancy, it seems reasonable to request a PCR for COVID-19, since it could be a rare presentation of SARS-CoV-2.

CoA Synthase (COASY) Mediates Radiation Resistance via PI3K Signaling in Rectal Cancer.

Neoadjuvant radiation is standard of care for locally advanced rectal cancer. Response to radiation is highly variable and directly linked with survival. However, there currently are no validated biomarkers or molecular targets to predict or improve radiation response, which would help develop personalized treatment and ideally targeted therapies. Here, we identified a novel biomarker, coenzyme A synthase (COASY), whose mRNA expression was consistently elevated in radioresistant human rectal cancers. This observation was validated in independent patient cohorts and further confirmed in colorectal cancer cell lines. Importantly, genetic overexpression and knockdown yielded radioresistant and sensitive phenotypes, respectively, in vitro and in vivo. COASY-knockdown xenografts were more vulnerable to radiation, showing delayed tumor growth, decreased proliferation, and increased apoptosis. Mechanistically, COASY protein directly interacted with the PI3K regulatory subunit PI3K-P85α, which increased AKT and mTOR phosphorylation, enhancing cell survival. Furthermore, shRNA COASY knockdown disrupted downstream PI3K pathway activation and also hindered DNA double-strand break repair, which both led to improved radiosensitivity. Collectively, this work reveals for the first time the biological relevance of COASY as a predictive rectal cancer biomarker for radiation response and offers mechanistic evidence to support COASY as a potential therapeutic target. SIGNIFICANCE: COASY is a novel radiotherapy response modulator in rectal cancer that regulates PI3K activation and DNA repair. Furthermore, COASY levels directly correlate with radiation response and serve as a predictive biomarker.

Expression of Sigma-Class Glutathione-S-Transferase in Fetal and Pediatric Filum Terminale Samples: A Comparative Study.

Background and objectives: The pathophysiology of tethered cord syndrome (TCS) in children is not well elucidated. An inelastic filum terminale (FT) is the main factor underlying the stretching of the spinal cord in TCS. Our study aimed to investigate the expression of glutathione-S-transferase (GST) in children and fetal FT samples in order to understand the relationship between this enzyme expression and the development of TCS. Materials and Methods: FT samples were obtained from ten children with TCS (Group 1) and histological and immunohistochemical examinations were performed. For comparison, FT samples from fifteen normal human fetuses (Group 2) were also analyzed using the same techniques. Statistical comparison was made using a Chi-square test. Results: Positive GST-sigma expression was detected in eight (80%) of 10 samples in Group 1. The positive GST-sigma expression was less frequent in nine (60%) of 15 samples from Group 2. No statistically significant difference was detected between the two groups (p = 0.197). Conclusions: Decreased FT elasticity in TCS may be associated with increased GST expression in FT. More prospective studies are needed to clarify the mechanism of the GST-TCS relationship in children.

Recent Progress in the Development of Fluorometric Chemosensors to Detect Enzymatic Activity.

Enzymes are a class of macromolecules that function as highly efficient and specific biological catalysts requiring only mild reaction conditions. Enzymes are essential to maintaining life activities, including promoting metabolism and homeostasis, and participating in a variety of physiological functions. Accordingly, enzymatic levels and activity are closely related to the health of the organism, where enzymatic dysfunctions often lead to corresponding diseases in the host. Due to this, diagnosis of certain diseases is based on the levels and activity of certain enzymes. Therefore, rapid real-time and accurate detection of enzymes in situ are important for diagnosis, monitoring, clinical treatment and pathological studies of disease. Fluorescent probes have unique advantages in terms of detecting enzymes, including being simple to use in highly sensitive and selective real-time rapid in-situ noninvasive and highly spatial resolution visual imaging. However, fluorescent probes are most commonly used to detect oxidoreductases, transferases and hydrolases due to the processes and types of enzyme reactions. This paper summarizes the application of fluorescent probes to detect these three types of enzymes over the past five years. In addition, we introduce the mechanisms underlying detection of these enzymes by their corresponding probes.

N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis.

The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

Serum biochemical and haematological reference intervals for water buffalo Bubalus bubalis heifers.

Based on a review of the literature, reference intervals for water buffalo (Bubalus bubalis) serum biochemistry and haematology have not previously been published. The current study was done to establish reference intervals for water buffalo heifers. The International Federation of Clinical Chemistry stated that at least 120 values are necessary to obtain reliable estimates for reference intervals. A total number of 127 clinically healthy buffalo heifers (1-2 years old) were included in the study. Animals were examined at buffalo farms that belong to Assiut Governorate, Egypt. Three types of samples were collected: serum samples for biochemical analysis, whole blood samples for haematological analysis and faecal samples for parasitological examination. Animals that fitted the inclusion criteria were included in the study. Biochemical analysis included serum total proteins, albumin, total globulins, alpha, beta and gamma globulin levels, and aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, creatine phosphokinase and lactate dehydrogenase activity. In addition to the above, serum creatinine, urea, total bilirubin, direct bilirubin, indirect bilirubin, sodium, potassium, chloride, magnesium, calcium, phosphorus, copper, zinc, iron, triglycerides, high density lipoprotein, low density lipoprotein, very low density lipoprotein, glucose levels and 20 haematological variables were measured. The 95.0% reference intervals were calculated by removing the upper and lower 2.5% of the interval for each serum biochemical constituent to give the 2.5 and 97.5 percentiles. Confidence intervals were calculated for each reference limit. Reference intervals from the current study were compared with established values for cows. The current study is as far as could be determined the first that establishes reference intervals for the serum biochemical and haematological parameters in water buffalo heifers.

Assessment of Escherichia coli O157:H7 transference from soil to iceberg lettuce via a contaminated field coring harvesting knife.

The potential for lettuce field-coring harvesting knives to cross-contaminate lettuce heads with pathogens was evaluated. Rings and blades of the harvest knives artificially contaminated with Escherichia coli O157:H7 (EHEC), were used to core three successive heads of iceberg lettuce. The coring rings and blades were inoculated by dipping into soils containing EHEC at concentration ranges of 1-10(5) MPN/g soil. Factors that influenced EHEC transference from soil to iceberg lettuce via contaminated coring knife blade, included water content (WC) of clay and sandy soils, EHEC concentration, and degree of blade contact (stem, medium, and heavy) with edible tissue. High moisture content clay soil was positively associated with high pathogen transference. No EHEC were detected on any cut heads when clay soil contaminated with 10(5) MPN/g EHEC had WC of 20% or less, or when the knife blade was dipped into sandy soil contaminated with EHEC at the same level, regardless of percent WC. The extent to which the harvesting knife blade cut across edible lettuce tissues was also an important factor in the amount of pathogen transference that occurred. EHEC were detectable on first and second sequentially cut lettuce heads when medium-contact was made between knife blade and edible tissues and on all three sequentially cut lettuce heads using the heavy-contact cutting scenario, when the blade was contaminated with 10(4) cfu/g EHEC in clay soil (25% WC). However, when the blade, contaminated at the same soil EHEC level, was used to cut only the stem and had no contact with the edible portion of the lettuce head, no pathogen transference was detected. Under the current CIF harvesting practice, the cutting blade has a higher potential than the coring ring to be contaminated by the soil, but less opportunity to transfer pathogens to harvested lettuce. However, once contaminated, the coring ring has much higher potential than the blade to transfer pathogens to the harvested lettuce.

Establishment of a simple detection system for blood group ABO-specific transferase activity in DNA-transfected cells.

A/B-transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen resulting in the synthesis of glycoproteins and glycolipids termed A/B-antigens. The ABO blood group (ABO) gene encoding A/B-transferase possesses numerous polymorphisms affecting the specificity and/or activity of the enzyme. The relationship between genotype and phenotype is very complicated, except for those of some critical polymorphisms. In order to establish a system for evaluating the effect of each polymorphism on the transferase function, an A- or B-transferase cDNA expressing vector was introduced into HeLa cells, a cell line that do not possess endogenous A/B-transferase activity. We successfully detected substrate-specific transferase activity in the cells and in the culture medium. Furthermore, in three different assays, each corresponding A- or B-antigen was detected in the transfectants with high sensitivity. Accordingly, the present study demonstrates a possibility that A/B-transferase variants may be characterized by using this method.

Analysis of 1-deoxy-D-xylulose 5-phosphate synthase activity in Grey poplar leaves using isotope ratio mass spectrometry.

Deoxy-xylulose phosphate synthase (DXS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway and it might regulate the metabolic flux in plastidic isoprenoid biosynthesis. We developed a sensitive assay suitable for plant extracts that is based on the decarboxylation of labeled pyruvate (1-(13)C)-PYR and detection of (13)CO(2) by isotope ratio mass spectrometry. We tested our method investigating the DXS activity in poplar leaves. Apparent DXS activity showed Michaelis constants of 111 and 158 microM for glyceraldehyde phosphate and pyruvate, respectively; pH and temperature optima were found at pH 8.6 and 45 degrees C. DXS activity was inhibited when the competitive inhibitor beta-fluoropyruvate was added to the reaction mixture. DXS activity strongly depended on leaf development with higher activity in young leaves and correlated fairly well with leaf isoprene emission potential. In mature poplar leaves, isoprene emission is the main metabolic sink of plastidic isoprenoid intermediates. Consequently, we found lower DXS activity in non-isoprene-emitting lines of poplar than in emitting plants as indicator of a lower demand of metabolic flux within the MEP pathway.

Mutagenesis of Aspergillus oryzae IPT-301 to improve the production of ¥â-fructofuranosidase

Aspergillus oryzae IPT-301, previously reported as a ¥â-fructofuranosidase producing microorganism, was successfully mutated using UV irradiation at 253.7 nm followed by the screening of survivors resistant to certain stress conditions. Strains were first subjected to the ¥â-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with fructofuranosidase activity values relative to the parent culture between 140 -190 percent were selected from survivors grown at temperature of 40¨¬C or 0.018 percent (w/v) sodium dodecyl sulfate concentration. They were cultivated on a rotary shaker to characterize mycelium and extracellular fructosyltransferase activities. Three mutants named IPT-745, IPT-746 and IPT-748 showed the highest amount of mycelium activity whose values increased 1.5 -1.8 fold, compared with the parent strain. It was found that more than 55 percent of total enzyme activity (mycelium- plus extracellular- activity) from these strains was detected in the mycelium fraction. Only one mutant, IPT-747, exceeded the amount of extracellular enzyme exhibited by the parent strain (1.5 times). This mutant also showed the highest value of total fructosyltransferase activity.

Determinación in vivo del papel de las enzimas esterasas y glutation transferasa en la resistencia a piretroides en Aedes aegypti (Diptera: Culicidae) / Determination in vivo of the role of esterase and gluthation transferase enzymes in pyrethroid resistance of Aedes aegypti (Diptera: Cullicidae)

Se realizó un estudio in vivo a través del uso de 2 sinergistas, el trifenil fosfato (TFF) inhibidor específico de esterasas y el ácido etacrínico (AE), inhibidor específico de la enzima glutation transferasa (GST), para determinar si estas enzimas eran responsables de la resistencia a piretroides en Aedes aegypti. Para el trabajo se utilizaron 2 cepas de Aedes aegypti resistentes a insecticidas, una cepa que fue seleccionada con temefos por 6 generaciones de selección (SAN-F6) y otra con deltametrina por 12 generaciones de selección con este insecticida (SAN-F12), ambas resultaron ser resistentes a insecticidas piretroides. Se demostró a través del uso de los sinergistas TFF y AE que las enzimas esterasas y GST son responsables de la resistencia a los piretroides en estas cepas. Esos resultados demuestran la existencia de un fenómeno de resistencia cruzada y multirresistencia, lo cual debe tenerse en cuenta en las estrategias de uso de insecticidas para el control de este vector.

An in vivo study of two synergists, that is, Triphenil phosphate -specific esterase inhibitor- and ethacrynic acid specific gluthation transferase inhibitor- was performed to determine if these enzymes were responsible for pyrethroid resistance of Aedes aegypti. To this end, two insecticide resistant Aedes aegypti strains were used, one strain selected with temephos by six selection generations (SAN-F6) and the other strain with delmamethrin by 12 selection generations (SAN-F12), being both strains resistant to pyrethroid insecticices. Through the use of TPP and EA synergists, it was proved that esterase and gluthation-s-transferase (GST) enzymes were responsible for pryrethroid resistance of these strains. These results showed the existence of cross-resistance and multidrug resistance, which should be taken into account for insecticide use strategies aimed at vector control.