Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.     

Recent increase of Marek's disease in Korea related to the virulence increase of the virus

The incidence of Marek's disease (MD), an important neoplastic disease of chickens, suddenly increased in 1997 in Korea. Most MD cases of this country were detected in chickens over 20 wk of age. Five MD viruses were isolated from field flocks in which severe MD losses had occurred, and one of the viruses was studied to compare its pathotype with the prototype JM strain. The isolate KOMD-IC induced severe depression not only in body weight but also in relative bursal weight, and the depression by KOMD-IC was more severe than that induced by JM strain. In addition, the incidence of MD tumor caused by KOMD-IC was higher than that caused by the JM strain. The protective capacity of several MD vaccines was studied against challenge with KOMD-IC. The protective levels of several MD vaccines such as herpesvirus of turkeys (HVT), HVT plus SB1, and Rispens were usually lower against challenge with KOMD-IC than those challenged with JM strain, even if the chickens vaccinated with serotype 1 were not completely protected against challenge with KOMD-IC. The above results indicate that the virulence of KOMD-IC isolated recently was increased, and the increase of MD outbreak in Korea may be related to the virulence increase of the virus. Various MD vaccine programs were applied to reduce MD loss to a broiler breeder farm where severe MD loss had occurred. Serotype 1 vaccine could dramatically decrease the mortality due to MD, and the best results were obtained from the flocks vaccinated with bivalent vaccine of Rispens and HVT.     

Changes in glial fibrillary acidic protein immunoreactivity in the dentate gyrus and hippocampus proper of adult and aged dogs

Astrocytes perform neuron-supportive tasks, repair and scarring process in the central nervous system. In this study, we observed glial fibrillary acidic protein (GFAP), a marker for astrocytes, immunoreactivity in the dentate gyrus and hippocampus proper (CA1-3 region) of adult (2-3 years of age) and aged (10-12 years of age) dogs. In the adult group, GFAP immunoreactive astrocytes were distributed in all layers of the dentate gyrus and CA1-3 region, except in the stratum pyramidale of the CA1-3 region. In the aged group, GFAP immunoreactivity decreased markedly in the molecular layer of the dentate gyrus. However, GFAP immunoreactivity in the CA1-3 region increased in all layers, and the cytoplasm of GFAP immunoreactive astrocytes was hypertrophied. GFAP protein levels in the aged dentate gyrus decreased; however, GFAP levels in the CA1-3 region increased. These results suggest that the morphology of astrocytes and GFAP protein levels in the hippocampal dentate gyrus and CA1 region are changed, respectively, with age.     

Antioxidant properties of monomeric,oligomeric, and polymeric fractions in hot water extract from <Emphasis Type="Italic">Pinus radiata</Emphasis> bark

Hot water extract (HWE) from Pinus radiata bark was fractionated into monomeric polyphenol (MPP), oligomeric proanthocyanidin (OPA), and polymeric proanthocyanidin (PPA) fractions by monitoring the UV-visible spectrum of the eluted fractions. Nine polyphenols were identified in MPP: three phenolic acids and six flavonoids. The major compounds of MPP were taxifolin (456 μg/mg), catechins (240 μg/mg), and protocatechuic acid (46.2 μg/mg). The OPA components ranged from dimers to hexamers. PPA comprised procyanidin (PC, 94%), a small amount of prodelphinidin (PD, 6%), and also the higher trans configuration, rather than the cis configuration, of C2-C3. The average molecular weight (Mw, Mn), polydispersity (Mw/Mn), and DP of PPA were 3,800 (Mw), 1,200 (Mn), 3.2 and 13, respectively. The potential antioxidant activities of HWE and the three fractions were estimated using proton- or electron-donating assays containing 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical cation, superoxide anion (O₂^•−), hydrogen peroxide (H₂O₂), reducing power and lipid peroxidation. All the bark fractions exhibited potent quenching abilities against both commercially available radicals (DPPH•, ABTS^•+) and chemically induced radicals (H₂O₂, O₂^•−), as well as strong reducing power and inhibitory activity of lipid peroxidation. Particularly, OPA showed a hydrogen peroxide scavenging activity significantly higher (p < 0.05) than that of the other fractions. From 2-thiobarbituric acid reactive substances and ferric thiocyanate assays, PPA was suspected to act by essentially delaying the formation of hydroperoxide by quenching free oxygen radicals that operates in oil-in-water emulsion.     

Adsorption of metal and uranyl ions onto amidoximated Pinus densiflora bark

Pine (Pinus densiflora) bark was chemically modified by grafting with acrylonitrile (AN) through Fenton reaction and conversion of the cyano group to an amidoxime group by reaction with hydroxylamine. Treatments of pine bark with 160 mM H₂O₂ at 40°C yielded ~70% grafting in 6-h reaction. After alkali hydrolysis of amidoximated pine bark (AOPB), its adsorption capacity (q _e) for copper ions was largely increased from 0.5 to 1.8 mmol/g. Adsorption of the hydrolyzed AOPB (HAOPB) followed the pseudo-second-order kinetic model. Among the metal ions tested, the order of adsorption capacity (q _e) predicted by the kinetic model at equilibrium time was Hg²⁺ > UO₂ ²⁺ > Cd²⁺ > Cu²⁺ > Zn²⁺ > Ni²⁺. The adsorption process was pH-dependent, and the adsorption equilibrium was observed in the pH range of 6–7 for all the metal ions. Even though pine bark is a good adsorbent for Hg²⁺ and UO₂ ²⁺ ions, it had a poor affinity for the other ions tested. It was obviously found that HAOPB had very high adsorption abilities for heavy metal and uranyl ions.     

Use of clomipramine,alprazolam, and behavior modification for treatment of storm phobia in dogs

OBJECTIVE: To evaluate use of clomipramine, alprazolam, and behavior modification for treatment of storm phobia in dogs. DESIGN: Prospective open clinical trial. ANIMALS: 40 dogs with storm phobia. PROCEDURE: Dogs received clomipramine at a dosage of 2 mg/kg (0.9 mg/lb), PO, every 12 hours for 3 months; then 1 mg/kg (0.45 mg/lb), PO, every 12 hours for 2 weeks; then 0.5 mg/kg (0.23 mg/lb), PO, every 12 hours for 2 weeks. Alprazolam was given at a dosage of 0.02 mg/kg (0.009 mg/lb), PO, as needed 1 hour before anticipated storms and every 4 hours as needed. Desensitization and counter-conditioning were conducted at home by the caregiver with an audio simulation of storm sounds that had induced a fear response during evaluation. RESULTS: 30 of the 32 dogs that completed the study had a degree of improvement, as measured by caregivers' global assessment. Two caregivers considered the storm phobia to be resolved. Panting, pacing, trembling, remaining near the caregiver, hiding, excessive salivation, destructiveness, excessive vocalization, self-trauma, and inappropriate elimination all decreased significantly during treatment. Improvement was greater during true storms (rain, thunder, and lightning) than during rain only. Response to audio simulation did not change during treatment. Four months after the study, improvement was maintained. CONCLUSIONS AND CLINICAL RELEVANCE: The combination of clomipramine, alprazolam, and behavior modification can be effective in decreasing or eliminating storm phobia. Improvement could not be evaluated by use of audio simulation of a storm.     

Eco‐efficiency as a strategy for optimizing the sustainability of pest management

Agricultural industrialization and the subsequent reliance on pesticides has resulted in numerous unintended consequences, such as impacts upon the environment and by extension human health. Eco‐efficiency is a strategy for sustainably increasing production, while simultaneously decreasing these externalities on ecological systems. Eco‐efficiency is defined as the ratio of production to environmental impacts. It has been widely adopted to improve chemical production, but we investigate the challenges of applying eco‐efficiency to pesticide use. Eco‐efficiency strategies include technological innovation, investment in research and development, improvement of business processes, and accounting for market forces. These components are often part of integrated pest management (IPM) systems that include alternatives to pesticides, but its implementation is often thwarted by commercial realities and technical challenges. We propose the creation and adoption of an eco‐efficiency index for pesticide use so that the broad benefits of eco‐efficient strategies such as IPM can be more readily quantified. We propose an index based upon the ratio of crop yield to a risk quotient (RQ) calculated from pesticide toxicity. Eco‐efficiency is an operational basis for optimizing pest management for sustainability. It naturally favors adoption of IPM and should be considered by regulators, researchers, and practitioners involved in pest management. © 2019 Society of Chemical Industry     

Investigation of the prevalence of neurologic equine herpes virus type 1 (EHV-1) in a 23-year retrospective analysis (1984–2007)

A single nucleotide polymorphism in the equine herpesvirus 1 (EHV-1) DNA polymerase gene (ORF30 A₂₂₅₄ to G) has been associated with clinical signs of equine herpes myeloencephalopathy (EHM). The purpose of our study was to determine the odds ratio for this genetic marker and EHM using a panel of field isolates from North America collected over the past twenty-three years. EHV-1 isolates cultured at the Cornell University Animal Health Diagnostic Laboratory from 1984 to 2007 were retrieved along with their clinical histories. DNA was extracted from these EHV-1 cultures and allelic discrimination was performed using real-time PCR. The results were confirmed by sequencing of the target region in ORF30. PCR and sequencing were in 100% agreement and showed that 19 out of the 176 isolates had the ORF30 G₂₂₅₄ allele (11%), of which16 were EHM cases and 3 respiratory or abortion cases. The odds of having neurologic disease with the ORF30 G₂₂₅₄ genotype were computed as 162 times greater than those with the opposite allele ORF30 A₂₂₅₄ (95% confidence interval: 35–742). Despite this strong statistical significance, 24% (5/21) of horses with neurologic disease in our study population harbored the “non-neurologic” form of the allele (ORF30 A₂₂₅₄), suggesting that other factors may also contribute to the onset of EHM.     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.