Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.

Light-accelerated "on-water" hydroacylation of dialkyl azodicarboxylates.

The hydroacylation of dialkyl azodicarboxylates has received a lot of attention lately due to the great importance of acyl hydrazides in organic chemistry. Herein, we report an inexpensive and green photochemical approach, where light irradiation (390 nm) significantly accelerates the reaction between dialkyl azodicarboxylates and aldehydes, while water is employed as the solvent. A variety of aromatic and aliphatic aldehydes were converted into their corresponding acyl hydrazides in good to excellent yields in really short reaction times (15-210 min) and the reaction mechanism was also studied. Applications of this reaction in the syntheses of Vorinostat and Moclobemide were demonstrated.

Cyrene: a bio-based solvent for the Mizoroki-Heck reaction of aryl iodides.

The development of greener and more sustainable methods, as well as the adaptation of already existing protocols to more environmentally friendly procedures, has become crucial for organic synthesis. The introduction and utilization of greener solvents is a very promising alternative, especially when they can replace toxic organic solvents in the known and widely used organic reactions. Cyrene has appeared to be an excellent alternative solvent for a number of organic reactions. In this work, the development of a new, greener and more economical protocol for the Mizoroki-Heck reaction is described, using Cyrene as the green solvent and Pd/C as the palladium catalyst source. A wide substrate scope for the coupling of aryl iodides with acrylamides, acrylates, acrylic acid, acrylonitrile and styrene was demonstrated. The recyclability of Cyrene and the leaching of palladium in the final product were examined in order to enhance the industrial applicability of this protocol. Furthermore, the synthesis of the natural product piperlotine A is reported.

Coal combustion residues and their effects on trace element accumulation and health indices of eastern mud turtles (Kinosternon subrubrum).

Coal combustion is a major energy source in the US. The solid waste product of coal combustion, coal combustion residue (CCR), contains potentially toxic trace elements. Before 1980, the US primarily disposed of CCR in aquatic settling basins. Animals use these basins as habitat and can be exposed to CCR, potentially affecting their physiology. To investigate the effects of CCR on eastern mud turtles (Kinosternon subrubrum), we sampled 30 turtles exposed to CCRs and 17 unexposed turtles captured in 2015-2016 from the Savannah River Site (Aiken, SC, USA). For captured turtles, we (1) quantified accumulation of CCR in claw and blood samples, (2) used bacterial killing assays to assess influences of CCR on immune responses, (3) compared hemogregarine parasite loads, and (4) compared metabolic rates via flow-through respirometry between CCR-exposed and unexposed turtles when increased temperature was introduced as an added stressor. Turtles exposed to CCR accumulated CCR-associated trace elements, corroborating previous studies. Blood Se and Sr levels and claw As, Se, and Sr levels were significantly higher in turtles from contaminated sites. Average bacterial killing efficiency was not significantly different between groups. Neither prevalence nor average parasite load significantly differed between CCR-exposed and reference turtles, although parasite load increased with turtle size. Regardless of site, temperature had a significant impact on turtle metabolic rates; as temperature increased, turtle metabolic rates increased. The effect of temperature on turtle metabolic rates was less pronounced for CCR-exposed turtles, which resulted in CCR-exposed turtles having lower metabolic rates than reference turtles at 30 and 35 °C. Our results demonstrate that turtles accumulate CCR from their environment and that accumulation of CCR is associated with changes in turtle physiological functions when additional stressors are present.

Distinctive roles of PKC delta isozyme in platelet function.

Platelet activation is a complex balance of positive and negative signaling pathways. Several protein kinase C (PKC) isoforms are expressed in human platelets. They are a major regulator of platelet granule secretion, activation and aggregation activity. One of those isoforms is the PKCδ isozyme, it has a central yet complex role in platelets such as opposite signaling functions depending on the nature of the agonist, it concentration and pathway. In fact, it has been shown that PKCδ has an overall negative influence on platelet function in response to collagen, while, following PAR stimulation, PKCδ has a positive effect on platelet function. Understanding the crucial role of PKCδ in platelet functions is recently emerging in the literature, therefore, further investigations should shed light into its specific role in hemostasis. In this review, we focus on the different roles of PKCδ in platelet activation, aggregation and thrombus formation.

PKCs in thrombus formation.

The protein kinase C (PKC) family has been implicated in several physiological processes regulating platelet activation. Each isoform of PKC expressed on platelets, may have a positive and/or negative role depending on the nature and concentration of the agonist. Mice lacking PKCα show much reduced thrombus formation in vivo, while PKCθ(-/-) showed inhibition of aggregation in response to PAR4. On the other hand, PKCδ by associating with Fyn, inhibits platelet aggregation. In addition, PKCß by interacting with its receptor RACK1 has been implicated in the primary phases of signaling via the αIIbß3 and finally PKCɛ appears to be involved in platelet function downstream GPVI. The present review discusses the latest observations relevant to the role of individual PKC isoforms in platelet activation and thrombus formation.

Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis).

Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (Km) for dihydroxyacetone is 137.39 (+/- 25.56) microM whereas the Km for glycerol-3-phosphate is 468.66 (+/- 27.59) microM. The kinetic mechanism of purified enzyme is 'ordered Bi-Bi' and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35 degrees C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (+/- 1000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (+/- 2000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mM N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn2+ and by 2,4-dichlorophenoxyacetic acid.

Discovery of a novel CCR3 selective antagonist.

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.

Design, synthesis, and discovery of a novel CCR1 antagonist.

The CC chemokines may play an important role in the pathogenesis of chronic inflammatory diseases including rheumatoid arthritis, and their effects are thought to be mediated through CCR1 receptors. Several nonpeptide CCR1 receptor antagonists that showed high affinity for human CCR1 receptors have been identified; however, their effectiveness in animal models of inflammatory diseases has been scarcely demonstrated, probably due to species selectivity of the antagonists. To elucidate the pathophysiological role of CCR1 receptors in murine models of disease, we looked for a potent antagonist for both murine and human CCR1 receptors. Screening of our chemical collection for inhibition of (125)I-MIP-1alpha binding to human CCR1 receptors transfected in CHO cells led to the identification of xanthene-9-carboxamide 1a as the lead compound. Derivatization of 1a by quaternarizing the piperidine nitrogen with various alkyl groups and by installing substituents into the xanthene moiety dramatically improved the inhibitory activity against both human and murine CCR1 receptors. As a result, 2q-1 showing IC(50) values of 0.9 and 5.8 nM for human and murine CCR1 receptors, respectively, was discovered. This compound is the first murine CCR1 receptor antagonist and may be a useful tool for clarifying the role of CCR1 receptors in murine models of disease.

Identification of a potent and nonpeptidyl ccr3 antagonist.

CCR3 is expressed in a variety of leukocyte subsets, especially eosinophils, and may be involved in allergic disorders such as atopic asthma. To clarify the pathophysiological roles of CCR3 in allergic disorders, we developed a nonpeptidyl CCR3 antagonist. This antagonist, which is referred to as "Compound X," that inhibited the binding of [(125)I]Eotaxin to CHO cells transfected with human CCR3 with an IC(50) value of 2.3 nM. In human eosinophils, Compound X also inhibited Eotaxin-induced increases in intracellular Ca(2+) concentrations and chemotaxis. Thus, Compound X appears to be a highly potent CCR3 antagonist. These findings suggest that Compound X may be a useful tool for elucidating the pathophysiological roles of CCR3 in a variety of allergic disorders.

Effect of hibernation, thyroid hormones and dexamethasone on cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase from jerboa (Jaculus orientalis).

Tissue distribution of the cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) activities in jerboa (Jaculus orientalis), a hibernator, shows the highest level of enzyme activity in skeletal muscle and brown adipose tissue, respectively. The effect of hibernation on cGPDH indicates an increase of activity in all tissues examined. In contrast, hibernation decreases mGPDH activity in all tissues, except skeletal muscle. The effect of thyroid hormones on GPDH activity was tissue specific: in kidneys, cGPDH activity doubled in euthermic jerboas treated with T4. In contrast, 6-n-propyl-2-thiouracil treatment provokes an increase of enzyme activity in brown adipose tissue, liver and brain. T4 treatment leads to a 2.7-fold increase in liver mGPDH activity. 6-n-propyl-2-thiouracil treatment decreases mGPDH activity in the skeletal muscle whereas the opposite effect was observed in brain. Dexamethasone stimulates cGPDH in all tissues examined, except skeletal muscle and kidneys. In the case of mGPDH activity, this increase was observed only for brown adipose tissue and brain. Our results suggest that hibernation, thyroid hormones and dexamethasone probably play a role in the regulation of cGPDH and mGPDH activities in jerboa. Our findings confirm that these enzymes are involved in metabolic adaptation to thermal stress in Jaculus orientalis.

Solanapyrones, Phytotoxins Produced by Alternaria solani: Biosynthesis and Isolation of Minor Components.

Two minor components of solanapyrones D and E, phytotoxins from Alternaria solani, were isolated. The absolute stereochemistry of D and the optical purity of solanapyrones A and D were determined by chemical transformation and measurement of the CD spectrum. In addition, complete signal assignment of the (1)H- and (13)C-NMR spectra of solanapyrones A and D, and their solution conformations were studied. Feeding experiments with various isotopically labeled acetates and methionine established the biosynthetic pathway of solanapyrones. The phytotoxicity of solanapyrones on lettuce seedlings is discussed.

[An evaluation of the attitudes and productivity of public health nurses in mental health service programs for the elderly].

The relationship between productivity of public health nurses (PHN) employed in mental health programs including home visit service for elderly people with dementia, and their attitudes toward mental health services was studied by surveying PHN at 31 health centers (HC) in Osaka Prefecture. A review of the services provided for 382 clients with dementia visited during a one-year period showed that PHN had provided the following services: home health care education: family education to promote understanding of the disease; management of interpersonal relations in the family; intervention to reduce the burden placed on the family members; medical facility referral; social service resource referral. For each of these PHN-provided services, the service had been provided to more than 60% of the clients. The percentage of clients who had been visited by a PHN accompanying a psychiatrist or a social worker was significantly higher at the 13 HC where PHN attach importance to counseling programs for the elderly, the 8 HC which provide group work services, and the 13 HC which hold case conferences more than six times a year than at other HC. In addition, the service provision rates for many PHN-provided services were significantly higher for these HC than for other HC. Analysis on the basis of whether PHN did or did not attach importance to the counseling program, showed that in either group, clients who had been visited by a PHN accompanying a psychiatrist or a social worker were more likely to have received PHN-provided services than other clients. Furthermore, at HC where PHN actively participated in mental health programs, PHN attitudes toward mental health care were more enthusiastic than at other HC. These results indicate that increased productivity of PHN employed in mental health programs and promotion of a multidisciplinary approach in client services are correlated with each other, and that both factors contribute to improving the quality of home visit services provided by PHN.