A polymorphism in XKR4 is significantly associated with serum prolactin concentrations in beef cows grazing tall fescue.

Fescue toxicosis is a common syndrome of poor growth and reproductive performance of beef cattle grazing endophyte-infected tall fescue infected with Lolium arundinaceum Schreb. Together with decreased feed intake, decreased growth rates and tissue necrosis due to vasoconstriction, depressed circulating serum prolactin concentrations are typically observed in cattle afflicted with fescue toxicosis. Polymorphisms within the XK, Kell blood group complex subunit-related family, member 4 (XKR4) gene located on BTA14 have been previously reported to be associated with rump fat thickness, residual feed intake, average daily feed intake and average daily gain in cattle. Associations also have been reported between XKR4 genotype and effectiveness of the dopamine antagonist iloperidone as a treatment of schizophrenia in humans. Domperidone, a related dopamine antagonist, mediates effects of fescue toxicosis on livestock, including restoring depressed concentrations of prolactin. A mixed-breed population of 592 beef cattle grazing endophyte-infected tall fescue was used to examine the association between XKR4 genotype and circulating prolactin concentrations. The SNP rs42646708 was significantly (P = 0.0002) associated with serum prolactin concentrations and explained 2.45% of the phenotypic variation. Effect of genotype at the SNP was tested across five breeds, with significant associations within both Angus (P = 0.0275) and Simmental (P = 0.0224) breeds. These results suggest XKR4 may play a role in mediating the negative effects of fescue toxicosis, and polymorphisms within this gene may be useful markers for selection for genetic resistance to the debilitating effects of endophyte-infected tall fescue.

Automatic carbon dioxide-methane gas sensor based on the solubility of gases in water.

Biogas methane content is a relevant variable in anaerobic digestion processing where knowledge of process kinetics or an early indicator of digester failure is needed. The contribution of this work is the development of a novel, simple and low cost automatic carbon dioxide-methane gas sensor based on the solubility of gases in water as the precursor of a sensor for biogas quality monitoring. The device described in this work was used for determining the composition of binary mixtures, such as carbon dioxide-methane, in the range of 0-100%. The design and implementation of a digital signal processor and control system into a low-cost Field Programmable Gate Array (FPGA) platform has permitted the successful application of data acquisition, data distribution and digital data processing, making the construction of a standalone carbon dioxide-methane gas sensor possible.

Gabapentin in horses: validation of an analytical method for gabapentin quantitation.

Gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin], is a new anticonvulsant used as adjunctive therapy in the treatment of partial seizures in humans not controlled with standard antiseizure drugs, and it has also been used in veterinary medicine. In performance horses, gabapentin is listed as a class 3 performance-enhancing substance by the Association of Racing Commissioners International, and thus is considered to have the potential to influence the outcome of races. Therefore, we developed and validated a sensitive gas chromatographic-mass spectrometric (GC-MS) method for gabapentin detection. Gamma-aminobutyric acid-d(2) (GABA-d(2)) was used as an internal standard during solid-phase extraction; lacking the cyclohexyl ring of gabapentin, GABA-d(2) formed a lactam structure to only a minor extent. Gabapentin, on the other hand, readily formed a lactam on thermal exposure during trimethylsilyl-derivatization and/or GC analysis; electrospray-ionization MS was employed to verify that the original compound was present as the expected 171 m.w. compound. Extraction efficiency for the assay was about 60%, and a curvilinear standard curve ranging from 50 ng/mL to 3000 ng/mL provided excellent within-run and between-run coefficients of variation and accuracies over a range of low, medium, and high values. The limit of detection, defined as the concentration calculated from the mean response at zero concentration plus two times the standard deviation, was calculated at 7.6 ng/mL; the limit of quantitation, defined as the concentration calculated from the mean of the zero responses plus five times the standard deviation, was calculated at 17 ng/mL. This method will enable accurate quantification of gabapentin in equine biological fluids for use in both pharmacokinetic and forensic studies.

Clonidine in horses: identification, detection, and clinical pharmacology.

Clonidine is classified as a class 3 performance-enhancing agent by the Association of Racing Commissioners International and thus has the potential to influence the outcome of a race. In this study, the authors developed and validated a sensitive gas chromatograph and mass spectrometer method to determine the pharmacokinetic parameters of clonidine in equine plasma samples after IV administration of a single dose (0.025 mg/kg) of clonidine in horses. At this dose, clonidine produced rapid and profound sedation, which cold be quickly reversed with yohimbine. Clonidine was able to produce an analgesic effect but failed to provide maximal analgesia in all horses; the limited analgesic effect persisted for about 60 minutes.

The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1.

The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices alpha2 and alpha3 of the BAG domain and predominantly residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (K(D) = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.

Core biopsy of the breast with atypical ductal hyperplasia: a probabilistic approach to reporting.

The diagnosis of atypical ductal hyperplasia (ADH) at needle core breast biopsy (NCB) is typically regarded as an indication for surgical excision. Although ADH is an intermediate risk nonobligate precursor lesion, the rationale for further therapy is the result of a reported high prevalence of a concomitant more advanced lesion (typically ductal carcinoma in situ) as the index lesion. To assess whether certain histopathologic features of ADH in NCB are predictive of open biopsy outcomes, the authors correlated the extent and pattern of ADH in 47 core biopsies (11-or 14-gauge) with the subsequent surgical specimen. Extent of ADH on NCB was ascertained by determining the number of large ducts and/or terminal duct-lobular units affected, with involvement of one large duct or one terminal duct-lobular unit representing a single focus, involvement of one duct and one terminal duct-lobular unit as two foci, and so on. Of the 47 cases, ADH was restricted to < or =2 foci in 24 cases (51.1%), confined to 3 foci in 8 cases (17.0%), and involved > or =4 foci in 15 cases (31.9%). The corresponding histopathologic findings at excision were benign lesions without atypia (n = 14), focal residual ADH (n = 13), atypical lobular hyperplasia (n = 3), ductal carcinoma in situ (n = 15), and invasive mammary carcinoma (n = 2). When the number of foci of involvement by ADH on NCB (based on an average of 11.6 cores per case) was correlated with the open biopsy results, all cases of ADH limited to < or =2 foci had no worse lesion on excision, whereas ADH present in > or =4 foci was found to be a strong predictor of a more advanced lesion on excision (p <0.0001, chi2). When histologic pattern was evaluated, all cases of pure micropapillary ADH on NCB showed pure micropapillary ductal carcinoma in situ on excision.

Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.

Symptomatic persistent post-coronary artery bypass graft pleural effusions requiring operative treatment : clinical and histologic features.

BACKGROUND: More than 85% of patients develop pleural effusions after coronary artery bypass grafting (CABG). Although the majority resolve spontaneously, post-CABG effusions can persist. The cause of these persistent effusions is unknown, and the histology of the pleural changes has seldom been reported. OBJECTIVES: To describe the patient characteristics and pathologic condition of the pleural tissues in patients with persistent post-CABG effusions. SUBJECTS: Eight patients with persistent post-CABG effusions who underwent thoracoscopy or thoracotomy over a 2-year period by one thoracic surgeon. These eight patients were selected as having undergone CABG > 2 months before their thoracic surgery and had no other identifiable causes of effusion. RESULTS: The median time from CABG to pleural surgery was 132 days (range, 74 to 2,258 days). The median left ventricular ejection fraction was 57% (range, 15 to 70%). All patients were dyspneic and had large (> or = 25% of the hemithorax) effusions on chest radiograph. All effusions persisted after two or more thoracenteses. Pleural effusion was left sided in three patients and bilateral in five patients. Pleural fluid was characterized by lymphocytosis (82 to 99%). Four of the eight patients had a visceral peel and trapped lung requiring decortication. Seven of the eight biopsy specimens showed pleural thickening characterized by dense fibrous tissues with associated mononuclear cell infiltration, while the eighth biopsy specimen showed only clotted blood. The degree of inflammation and fibrosis correlated with the interval between CABG and pleural surgery. Early post-CABG patients displayed more inflammation, with abundant lymphocytes in nodular configuration deep in the fibrous tissues away from the surface. Abundant keratin-positive, spindle-shaped cells were present in the fibrous tissues. Late cases showed predominantly mature fibrosis. CONCLUSIONS: Persistent post-CABG effusion can occur. Pleural fluids and pleural tissue in early-stage lesions were characterized by lymphocytosis. With time, the inflammatory changes were replaced by fibrosis that resulted in dyspnea and, at times, trapped lungs requiring surgical intervention.

Molecular basis for CD40 signaling mediated by TRAF3.

Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. This molecular adaptation may influence binding affinity and specific cellular triggers.

Ligand responsiveness in human prostate cancer: structural analysis of mutant androgen receptors from LNCaP and CWR22 tumors.

Androgen receptors (ARs) belong to the family of hormone receptors that are ligand-dependent transcription factors. Endocrine therapy provides effective treatment for prostate cancer until mutations arise that alter the ligand responsiveness of AR. In this study, structural models were developed for the functional domains of human AR by homology modeling from crystal structures of closely related nuclear receptors. These models were used to locate the sites of two frequently occurring mutations in prostate cancer. The substitutions that develop in LNCaP (threonine-->alanine at residue 877) and CWR22 (histidine-->tyrosine at residue 874) tumor cell lines are both located on helix 11 that forms part of the ligand-binding pocket. However, the results suggest that these mutations influence ligand responsiveness by completely different mechanisms. Residue 877 contacts the ligand directly, and substitution at this site alters the stereochemistry of the binding pocket. Thus, the LNCaP mutation apparently broadens the specificity of ligand recognition. In contrast, residue 874 is located down the helical axis, projects away from the ligand pocket, and does not contact ligand. The side chain of residue 874 lies in a cavity between helices 11 and 12. Substitution of tyrosine for histidine 874 in CWR22 tumors may affect a conformational change of helix 12 and, thus, influence binding of coactivator proteins and their regulatory effect on transcriptional activation.

Diabetic mastopathy. A clinicopathologic review.

Diabetic mastopathy, an uncommon form of lymphocytic mastitis and stromal fibrosis, typically occurs in longstanding type 1 diabetes. Nineteen cases meeting predetermined histopathologic criteria for diabetic mastopathy were correlated as to clinical history and disease recurrence. Physical examination revealed palpable discrete masses or diffuse nodularity, both predominantly in the subareolar region. One nonpalpable lesion was detected incidentally during reduction mammoplasty. All cases contained lymphocytic ductitis and lobulitis with varying degrees of keloidal fibrosis, vasculitis, epithelioid fibroblasts, and lymphoid nodule formation. Single mammary lesions were found in 11 patients with type 1 diabetes, 1 with type 2 diabetes, and 3 without diabetes. Four cases were bilateral (3 patients with type 1 and 1 patient with type 2 diabetes). Six of 19 cases recurred (3 ipsilateral, 2 contralateral, and 1 bilateral). We confirm the histopathologic constellation for diabetic mastopathy. However, we question the specificity of these features because of identical findings in patients with type 2 diabetes and nondiabetic patients. We found diabetic mastopathy in men and women, as a solitary mass or bilateral disease, and recurrence in either breast, sometimes multiple. Recognition of potential recurrence is important because it might spare patients with documented diabetic mastopathy from repeated breast biopsies.

A model for a glutamate receptor agonist antibody-binding site.

A combination of mutagenesis, computer modeling and immunoreactivity has been used to develop a structural model of a segment of the glutamate receptor (GluR), termed GluR3B, which is bound by receptor-activating autoantibodies. In this model, the GluR3B epitope is located in a reverse hairpin loop that places key residues important for antibody recognition and receptor activation in a linear arrangement on the solvent-exposed surface. The conformation of the loop is stabilized by a hydrophobic core which is critical for functional integrity of the epitope. The proximity of the amino- and carboxy-terminal residues suggested that the GluR3B peptide could be cyclized without diminishing immunoreactivity through replacement of these residues with cysteines and formation of a disulfide bond. This prediction was confirmed experimentally since the cyclized peptide retained full immunoreactivity. The model provides insight into GluR subunit-specific functional diversity and the role of autoantibodies to this region in neurological disease.

Differential requirements for tumor necrosis factor receptor-associated factor family proteins in CD40-mediated induction of NF-kappaB and Jun N-terminal kinase activation.

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-kappaB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-kappaB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Delta32), mediated NF-kappaB induction through a mechanism that was suppressible by co-expression of TRAF6(DeltaN), a dominant-negative version of TRAF6, but not by TRAF2(DeltaN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(DeltaN) did not impair JNK activation by CD40(Delta32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-kappaB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.

Interleukin-15 enhances host protection against acute Toxoplasma gondii infection in T-cell receptor alpha-/-deficient mice.

A potential role for T-cell receptor (TCR)-gamma(delta) cells in response to Toxoplasma gondii has been described. This study was undertaken to evaluate whether exogenous rIL-15 can enhance the production of gamma(delta) T cells in response to infection with T. gondii. For this, C57BL/6 and TCR alpha-gene deficient mice (alpha-/-) were vaccinated with Toxoplasma lysate antigen (TLA) in combination with interleukin (IL)-15. The cellular and humoral immune response was assessed following immunization. Administration of IL-15/TLA to alpha-/-mice lengthened survival time post lethal challenge. Several immunological parameters were increased in the alpha-/-mice following immunization with IL-15/TLA including serum immunoglobulin (Ig)G1 and IgG2a antibody titres and splenocyte lymphoproliferation in response to parasite antigen. Further, the CTL response to parasite infected target cells as well as the production of interferon gamma was enhanced by IL-15/TLA administration in the alpha-/-mice. These observations indicate that the gamma(delta) T cells response to this parasite can be enhanced by the administration of exogenous IL-15 in the presence of parasite lysate antigen.

Augmentation of the CD8+ T cell response by IFN-gamma in IL-12-deficient mice during Toxoplasma gondii infection.

The importance of IFN-gamma in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-gamma production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-gamma to parasite-infected p40-/- mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40-/- mice exhibit an increase in both precursor CTL frequency and IFN-gamma production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40-/- and IFN-gamma-/- mice. These observations indicate that IFN-gamma can regulate the CD8+ T cell response during T. gondii infection.

Conformational change in an anti-integrin antibody: structure of OPG2 Fab bound to a beta 3 peptide.

Antibodies are important tools to explore receptor-ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the alphaIIb beta3 integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the beta3 subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-A resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CL domains was observed so that the angle of rotation relating the two domains was reduced by 15 degrees. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment.

Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones.

The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, KD, of 100 nM. Kinetic analysis using surface plasmon resonance yielded a KD consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.