Color vision in horses (Equus caballus): Deficiencies identified using a pseudoisochromatic plate test.

In the past, equine color vision was tested with stimuli composed either of painted cards or photographic slides or through physiological testing using electroretinogram flicker photometry. Some studies produced similar results, but others did not, demonstrating that there was not yet a definitive answer regarding color vision in horses (Equus caballus). In this study, a pseudoisochromatic plate test--which is highly effective in testing color vision both in small children and in adult humans--was used for the first time on a nonhuman animal. Stimuli consisted of different colored dotted circles set against backgrounds of varying dots. The coloration of the circles corresponded to the visual capabilities of different types of color deficiencies (anomalous trichromacy and dichromacy). Four horses were tested on a 2-choice discrimination task. All horses successfully reached criterion for gray circles and demonstration circles. None of the horses were able to discriminate the protan-deutan plate or the individual protan or deutan plates. However, all were able to discriminate the tritan plate. The results suggest that horses are dichromats with color vision capabilities similar to those of humans with red-green color deficiencies. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Separation and characterization of mares' milk alpha(s1)-, beta-, kappa-caseins,gamma-casein-like,and proteose peptone component 5-like peptides

The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of alpha(s1)-CN primary structures of other species. In particular, 51, 48, 43, and 40% identities were obtained with corresponding regions of sow, dromedary, cow, and human alpha(s1)-CN, respectively. On the other hand, trace amounts of equine gamma-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to beta-CN peptides generated by plasmin action on the whole CN. The equine alpha(s1), beta-, and kappa-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric points distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The beta- and kappa-CN displayed a more acidic character in the mare than in the cow.     

Noscapine Acts as a Protease Inhibitor of In Vitro Elastase-Induced Collagen Deposition in Equine Endometrium

Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain (COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare’s endometrium and facilitate the therapeutic approach.     

Characteristic properties of equine milk proteins

Summary Properties of three equine milk proteins, lysozyme, α-lactalbumin, and β-lactoglobulin, were investigated and compared with the corresponding ones of bovine milk. The subsite structure similar to that in hen egg lysozyme was assumed in the functional region of lysozyme from interaction with inhibitors. α-lactalbumin has structures similar to those of bovine, but less stability of its intact and 3SS forms are observed than the corresponding bovine irrespective of [Ca²⁺] because of the entropy factors. Although the folding of β-lactoglobulin at acid pH is very similar to that of the bovine protein the conformation of the I strand is different from that of bovine I strand at neutral pH according to NMR data.     

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.     

Two-dimensional cartography of equine β-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction.     

液相色谱-串联质谱法测定马饲料中的抗凝血类药物

建立了马复合饲料中双香豆素、香豆素、醋硝香豆素、苄丙酮香豆素、苯茚二酮5种抗凝血类药物的高效液相色谱-串联质谱(HPLC-MS/MS)同时快速筛查和定量测定方法,并对该类化合物在电喷雾电离条件下的主要碎裂途径进行了探讨。方法以甲醇和0.02 mol/L NaOH溶液作为提取溶剂,饲料样品经提取后利用混合型阳离子交换固相萃取进行净化,采用正负切换多反应监测（MRM）模式对试样中5种香豆素类及茚二酮类抗凝血剂进行定性与定量分析。苯茚二酮的检出限为15 ng/g,香豆素和醋硝香豆素的检出限为4 ng/g,华法林和双香豆素的检出限为10 ng/g。5种药物的定量限在10~40 ng/g之间,日内日间精密度分别不高于8.63%和10.20%。     

东方马脑炎病毒E2蛋白的表达、纯化及免疫原性

目的表达、纯化东方马脑炎病毒(eastern equine encephalitis virus,EEEV)E2蛋白,并检测其对小鼠的免疫原性。方法利用IPTG诱导重组大肠埃希菌BL21-pET30-EEEV-E2,表达E2蛋白,用包涵体纯化试剂盒纯化重组E2蛋白,进行SDS-PAGE和Western blot分析。将BALB/c小鼠随机分为4组:PBS对照组、弗氏佐剂对照组(弗氏佐剂与PBS按体积比1∶1乳化)、E2蛋白组(E2蛋白与PBS按体积比1∶1混合)和E2蛋白+弗氏佐剂组(E2蛋白与弗氏佐剂按体积比1∶1乳化),每组10只,各组均经小鼠后肢肌肉免疫3次,两次免疫间隔时间均为14 d,免疫剂量均为100μl/只。第2次免疫后第7天,采用流式细胞术检测小鼠体内CD4+和CD8+T细胞比例;第2次免疫后第14天,采用细胞因子ELISA定量试剂盒检测小鼠血清中IL-2、IL-4和IFNγ的含量;第3次免疫后第7天,采用MTT法检测小鼠淋巴细胞增殖情况;每次免疫后第14天,采用ELISA法检测小鼠血清中IgG抗体效价。结果表达的重组E2蛋白相对分子质量约为53 000,表达量为菌体总蛋白的26.3%;纯化的重组E2蛋白纯度达95%以上;表达和纯化的重组E2蛋白均可与鼠抗His标签单克隆抗体结合。与PBS对照组、弗氏佐剂对照组和E2蛋白组相比,E2蛋白+弗氏佐剂组小鼠体内CD4+与CD8+T细胞比值、血清中IL-2、IL-4和IFNγ浓度、体内淋巴细胞增殖指数均明显升高(P﹤0.01);小鼠初次免疫后即可产生E2蛋白IgG抗体,且随着免疫时间的延长,抗体效价逐渐上升,第3次免疫后第14天,抗体效价可达1∶320。结论表达并纯化了重组E2蛋白,其能使小鼠产生较强的免疫反应,为新型EEEV疫苗的研制提供了参考。