Non-genomic steroid receptors in the bovine ovary

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.     

Morphological,morphometric and histochemical characterization of the gastric mucosa of the camel (Camelus dromedarius)

Mucosal morphology, morphometry and mucin histochemistry of the stomach were studied in the one-humped camel. The lining of the stomach was divided into eight grossly identifiable regions. The first region was non-glandular, occupied the body of the first compartment of the stomach and constituted 53.2% of the gastric mucosa. The other seven regions were lined by a glandular mucosa. Histological, histochemical and morphometric investigations have shown that glandular mucosa comprises pseudo-cardiac, cardiac, fundic and pyloric regions. The pseudo-cardiac region was characterized by widely separated short tubular serous glands. It constituted 36.2% of the gastric mucosa; it extended over the entire lining of the second compartment and parts of the first and third compartments. The cardiac region was confined to the initial zone of the third compartment amidst the psuedo-cardiac region but contiguous with the distal end of the gastric groove. It constituted 3.4% of the gastric mucosa and was characterized by neutral and acid mucin positive glands. The fundic and pyloric regions occupied the distal distended part of the third compartment. The fundic region constituted 4.3% of the gastric mucosa. It was packed with typical fundic glands characterized by chief cells, parietal cells and acid mucin positive neck cells. The pyloric region constituted 2.9% of the gastric mucosa. Its glands were positive to acid mucins except for their bases that were positive to neutral mucins. Differences in volume densities of the mucosal components and reactivity of the surface epithelium and gastric pits to mucin stains were noted in the different regions of the stomach.     

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.     

Association of tightly spiraled bacterial infection and gastritis in pigs

Tightly spiral bacteria were observed only in the pyloric mucosa of 4 (8.0%) of 50 swine stomachs, mainly in the surface of epithelia, the gastric pits and the lumen of gastric glands. The presence of the spiral bacteria was significantly associated with chronic pyloric gastritis (p<0.05). Mean gastritis score of the bacteria-positive pyloric mucosa was 3.25 +/- 0.25, whereas that of the bacteria-negative pyloric mucosa was 2.37 +/- 0.12. Parakeratosis and hyperkeratosis were spontaneously seen in the mucosa layer of pars oesophagea, regardless of the bacterial infection. Marked infiltration of mononuclear cells and granulocytes were seen in the cardiac mucosa, regardless of the bacterial infection. Mean gastritis score of the bacteria-positive cardiac mucosa was 3.27 +/- 0.32, whereas that of the bacteria-negative cardiac mucosa was 2.84 +/- 0.13. There was no significant difference between the bacteria-positive and negative cardiac mucosa (p>0.05). Inflammatory response in the fundic mucosa was rare (gastritis score=0.75 +/- 0.08). The tightly spiraled bactera were not cultured with various culture media. These results suggest that the presence of tightly spiraled bacteria is associated with only the pyloric gastritis in pigs.     

Paenibacillus spp.BD3526金属蛋白酶对乳蛋白水解位点分析

为了比较具有凝乳功能的Paenibacillus spp.BD3526来源的金属蛋白酶与基因重组凝乳酶对乳蛋白水解位点的差异,采用BD3526金属蛋白酶和凝乳酶对αs1-酪蛋白(casein,CN)、αs2-CN、β-乳球蛋白(lactoglobulin,Lg)和κ-CN进行酶解,分别对不同时间的酶解产物采用高效液相色谱与四极杆飞行时间串联质谱(high performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry,HPLC-Q-TOF-MS/MS)进行分析.结果表明:BD3526金属蛋白酶与凝乳酶在对乳蛋白的水解位点上具有较高的相似性,前者对αs1-CN、αs2-CN、β-Lg和κ-CN水解能力较后者弱,对P1为K(Lys)、R(Arg)且P1'为T(Thr)、F(Phe)和Y(Tyr)间的肽键水解特异性很高,并主要水解K-T (Lys-Thr)、K-F (Lys-Phe)、R-F(Arg-Phe)、R-Y(Arg-Tyr)肽键,水解生成的肽具有血管紧张素转换酶(angiotensin converting enzyme,ACE)抑制、抗菌、免疫调节等功能.     

Stimulated pepsinogen secretion from dispersed abomasal glands exposed to Ostertagia species secretion

Carbachol and secretions from Ostertagia species parasites significantly (P less than 0.001) stimulated isolated preparations of dispersed gastric glands from bovine and ovine abomasal mucosa to secrete pepsinogen. Atropine reduced the response to both secretagogues. Live adult and larval stages of Ostertagia ostertagi and O circumcincta and homogenates of these parasites did not significantly (P greater than 0.05) increase pepsinogen production from bovine or ovine gland preparations.     

Histological studies of the occurrence of organisms shaped like Campylobacter in the abomasum of calves]

The mucosa of the abomasum is a strongly acidic and therefore germ poor, but not entirely germfree environment. Only in isolated areas of the fundic glands zone were straight rods in the foveolas, and even more seldom spiral shaped bacteria could be encountered. They had no relation to the inflammatory infiltrates. On the other hand, Campylobacter-like organisms were more often observed in the pyloric region and, preferably, in a narrow zone following the cutaneous mucosa of the omasum. These organisms appeared to have a different size (1.3 to 2.4 microns length and 0.4 to 0.8 microns width) and they occurred deep inside the glands in an almost pure fashion. Whereas they caused no visible reaction of the tissue in the narrow cardiac zone, their occurrence in the pyloric region was several times connected with neutrophilic-rich infiltration. It ought to be tested, whether Helicobacter are among these Campylobacter-like organisms, which cause a disease leading up to ulcerations in calves similar to gastritis B in humans.     

Postnatal Development of the Stomach in the Japanese Field Vole, Microtus montebelli

The postnatal development of the stomach in the Japanese field vole, Microtus montebelli , were studied with the light and scanning electron microscopes.
Three kinds of cells constituting the fundic glands, that is, mucous neck cells, parietal cells and chief cells were distinguished clearly by light microscopy to have been differentiated around 14 days old. At 4 days old, the structure of pyloric glands was similar to that of adult stage. The investigation on the development of esophageal sac, fundic stomach and pyloric stomach was made by measuring each area of the vertical section. The fundic stomach grew more rapidly than the esophageal sac and pyloric stomach. The ratio of esophageal sac to fundic stomach and pyloric stomach in the area was about 1.5: 2: 1 at 20 days old.
In the fundic glands, 5-hydroxytriptamine (5-HT)- and somatostatin-immunoreactive cells appeared first at 6 days old and at birth, respectively and in the pyloric glands, gastrin-, 5-HT- and somatostatin-immunoreactive cells appeared at 19th day fetus. Gastrin-immunoreactive cells showed a rapid increase in the pyloric glands after birth. The distribution and frequency of endocrine cells were similar to that of adult at about 20 days old.
The fimbria showed bank-like appearance at the beginning and thereafter they showed the process-like structure. The fimbria in both the appearance and the length at 60 days old was similar to that of adult stage.
As a whole, the stomach of the field vole seemed to became mature at 20 days old in the aspects of gastric endocrine cells and morphology of gastric wall, although the fimbria formation was not accomplished at the same period.     

微小隐孢子虫抗原CTL细胞表位预测及多表位基因的原核表达

应用多个服务器对微小隐孢子虫（Cryptosporidium parvum）候选疫苗抗原的氨基酸序列进行分析,预测可能存在的CD8＋细胞毒性T细胞（CD8＋cytotoxic T lymphocyte,CD8＋CTL）表位。从6种常见的免疫效果较好的候选疫苗抗原基因中选出10个分值较高的表位基因串联在一起,形成一条多表位基因,进行人工合成,表位之间用柔性氨基酸GGGGS或GPGPG链接,命名为CpCTL10。将该多表位基因重组入高效融合表达载体pET-28a（＋）,获得重组质粒pET-28a（＋）-CpCTL10,转化大肠杆菌BL21（DE3）进行诱导表达,表达产物进行SDS—PAGE、Western blot分析。结果显示,CpCTL10基因在大肠杆菌中主要以包涵体形式高效表达,表达的重组蛋白占菌体蛋白总量的55．3％,纯化的重组蛋白纯度达75．1％。Western blot分析显示表达产物能与感染隐孢子虫的鼠阳性血清发生特异性反应,表明表达的重组蛋白具有较好的抗原性,为多抗原多表位疫苗的研制打下某础．     

The distribution of pepsinogen within the abomasa of cattle and sheep infected with Ostertagia spp. and sheep infected with Haemonchus contortus.

The effect of nematode infections on the production of pepsinogen by ruminants was investigated immunohistochemically and biochemically. Abomasal tissues were collected from parasite-naive cattle and sheep, from sheep infected with predominantly Ostertagia circumcincta, sheep infected experimentally with Haemonchus contortus and cattle infected with Ostertagia ostertagi. Pepsinogen was also assayed biochemically in homogenates of fundic mucosae from sheep infected with predominantly O. circumcincta. Infection with Ostertagia spp. parasites was associated mainly with nodular hyperplasia, resulting in increased numbers of cells that produce both pepsinogen and mucus. Measured biochemically, nodules contained more pepsinogen than adjacent more normal mucosa (p < 0.05), and this effect was largely attributable to the greater mass of nodules. Infection of sheep with H. contortus was associated with generalised hyperplasia, characterised by increased numbers of mucopeptic cells and in at least one animal with reductions in parietal cell numbers. At the same time, the zymogen granule content of chief cells was reduced. Similar changes were occasionally seen in sheep infected predominantly with O. circumcincta. Generalised hyperplasia is likely to be indicative of the presence of ambulatory parasitic stages as opposed to those confined to nodules. The potential for the enhanced production of pepsinogen by increased numbers of cells with a joint mucous cell and zymogenic cell phenotype may offset decreases in the numbers of chief cells or reductions in chief cell activity.     

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.     

Detection of antigenic variation among strains of Mycoplasma gallisepticum by enzyme-linked immunosorbent inhibition assay (ELISIA) and Western blot analysis

Polyclonal antisera (PCA) to three Mycoplasma gallisepticum (MG) strains (F, S6, and A5969) produced in rabbits were used in enzyme-linked immunosorbent inhibition assay (ELISIA) and Western blot analysis to examine antigenic variability among these strains of MG. In ELISIA, inhibiting antigen of the same strain used for immunization always led to the greatest percentage inhibition of PCA reactivity. Western blot analysis of antigens of four MG strains demonstrated both common and restricted patterns of immune recognition among the three PCAs.     

应用生物技术对奶牛进行基因改造的现状及展望

自从在乳品工业中使用可调控的凝乳酶重组体和牛生长激素后,基因操作已成为食品和农业生物工程学的前沿技术。早期对奶牛应用基因操作,目的是在牛奶中产生用于制药和医学的异种蛋白,这些应用为哺乳动物蛋白表达方面提供了先进的技术和经验,有助于对食物中的奶制品及奶蛋白进行遗传修饰。目前奶牛基因操作的研究领域主要还包括基因组定位、代谢途径、生长和发育、奶牛与微生物的相互作用。作者总结了这些领域的研究现状,综合一些技术、经济上的困难点和牛自身生殖系统上的限制对应用基因操作对奶牛进行基因改造进行了展望。     

Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test). The S. cruzi antibody-blocked Western blot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.     

Histology and Mucin Histochemistry of The Digestive Tract of Yellow Catfish, Pelteobagrus fulvidraco

The histology and characteristics of mucins secreted by epithelial mucous cells of the digestive tract in yellow catfish, Pelteobagrus fulvidraco were investigated using light microscope and transmission electron microscope. The digestive tract was divided into a pharynx, oesophagus, U-shaped stomach (with a cardiac, fundic and pyloric part) and intestine, composed of anterior intestine, middle intestine and posterior intestine, which consisted of a mucosa (epithelial layer), lamina propria-submucosa, muscularis and serosa. A large number of isolated longitudinal striated muscular bundles were present in the lamina propria-submucosa of pharynx. Goblet cells were observed throughout the digestive tract, except in the stomach. In the cardiac and fundic stomach, a plenty of gastric glands were observed, whereas they were absent in the pyloric part. Numerous mitochondria and endoplasmic reticulum were observed in the columnar epithelial cells of the intestine, especially of the anterior part. The epithelial mucous cells contained neutral or other two mixtures of acid and neutral mucins, the first being the most common. The neutral mucin was the only type of mucins in the stomach, anterior intestine and middle intestine. The results of this study will be helpful for understanding the digestive physiology and diagnosing some gastrointestinal diseases in yellow catfish.     

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.     

秦岭细鳞鲑消化系统的形态学观察

运用大体解剖和常规组织学方法对秦岭细鳞鲑消化系统的组织形态进行了系统观察。结果表明,秦岭细鳞鲑的消化道由口咽腔、食道、胃和肠等4部分组成,除口咽腔外,管壁由内向外分为黏膜、黏膜下层、肌层和浆膜。口咽腔宽大,上颌、犁骨及腭骨等处布有细齿,黏膜衬以复层扁平上皮,间有较多的黏液细胞和少量的杯状细胞及味蕾;食管粗而短,黏膜向管腔突出形成多个纵行皱襞,上皮由复层扁平上皮逐渐向单层柱状过渡,上皮细胞间可见到数量较多的粘液细胞和杯状细胞,食管腺不发达;胃体积较大,呈“U”形,包括贲门部、胃体和幽门部,胃体部小凹及胃底腺结构明显,肌层发达;胃与肠相接处有63-65个幽门盲囊,肠道粗短而略微盘曲,黏膜上皮为单层柱状,未见明显的肠腺。以上结果显示,秦岭细鳞鲑消化道组织结构与其肉食性功能密切相关。肝小叶界限不清;双列肝细胞围绕中央静脉呈放射状走行,肝细胞个体大、呈多边形,核呈圆形或卵圆形,1-2个。胰脏大部分环绕前肠边缘、呈细长条索状,另可见分散于幽门盲囊及胃周围的弥散部分;其外分泌部为管泡状腺,腺细胞呈锥体形,胰岛结构明显。     

Purification and partial characterization of canine pepsinogen A and B

OBJECTIVE: To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa. SAMPLE POPULATION: Stomachs obtained from 6 euthanatized dogs. PROCEDURE: Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, size-exclusion chromatography, and strong anion-exchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing. RESULTS: Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IER Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0.The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species. CONCLUSIONS AND CLINICAL RELEVANCE: Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs.     

Molecular characterization of Theileria orientalis piroplasm protein encoded by an open reading frame (To ORF2) in a genomic fragment

In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 bp genomic fragment amplified by PCR contained two open reading frames (ORFs). The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (To ORF2) showed homology to several surface antigens of plasmodia. To ORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-To ORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Indirect immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that To ORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic To ORF2 protein could be exploited for vaccine development against bovine piroplasmosis.