Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury

Radioimmunoassay and immunocytochemistry were used to study the distribution of galanin, a novel 29 amino acid porcine intestinal peptide, in the central nervous system of the rat and pig. The pattern of distribution was similar in the two species, with the highest concentrations of galanin-like immunoreactivity found in the neurohypophysis, hypothalamus and sacral spinal cord. Immunocytochemical studies of these regions localized galanin-like immunoreactivity to cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus, to fibres in the pars nervosa and to numerous cell bodies and fibres in the dorsal horn of the spinal cord. On both gel and high pressure liquid chromatography, galanin-like immunoreactivity in rat and pig nervous tissue eluted as a single peak in a position similar to purified procine intestinal galanin standard. Surgical and pharmacological manipulations in the rat suggest the presence of galanin in afferent fibres. An increase of galanin-like immunoreactivity was observed in the sacral spinal cord of the rat following thoracic spinal cord transection. Thus galanin-like immunoreactivity in the brain is mainly localized in the hypothalamopituitary region. The decrease of galanin-like immunoreactivity in the dorsal horn of the spinal cord, following dorsal rhizotomy and pre-treatment of rats with capsaicin, indicates that many of the fibres, which are of small diameter, may well be derived from spinal sensory neurones.     

Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni

The major phospholipids present in the phospholipid extract of Schistosoma mansoni were phosphatidylcholine (28%), phosphatidylethanolamine (25%), phosphatidylserine (15%) and phosphatidylglycerol (8%). The synthesis of phosphatidylcholine in S. mansoni adults occurred by the choline to phosphatidylcholine or Kennedy pathway. Incorporation of CDPcholine and choline into the phosphatidylcholine of worm slices appeared linear over time with no demonstrable sex differences in choline incorporation. A slight difference in the incorporation of CDPcholine by separate sexes was evident. Methylation of phosphatidylethanolamine to phosphatidylcholine could not be demonstrated.     

高效凝胶色谱一步法制备级纯化单克隆抗体

作者采用ＰｈａｒｍａｃｉａＳｅｐｈａｃｒｙｌＳ—３００凝胶色谱柱，建立了ＩｇＧ类ＭｃＡｂ的一步法制各级纯化方法。该法是将ＭｃＡｂ腹水直接上样，用ｐＨ７．４１０ｍｍｏｌ／ＬＰＢＳ洗脱（流速０．５ｍｌ／ｍｉｎ），即得到纯化的ＭｃＡｂ。一次上样量４０～５０ｍｌ腹水，回收率为８５％－９０％．整个纯化周期４ｈ。纯化的ＭｃＡｂ经ＳＤＳ—ＰＡＧＥ测定，纯度＞９０％，免疫组化ＡＢＣ法测定活性为１：８００００（７．８×１０－１１ｍｏｌ／Ｌ）。该法操作简单、快速，只要有一台核酸／蛋白检测仪，便可进行制备级水平的纯化。     

非受体型酪氨酸激酶-Syk蛋白的提取与纯化

目的 :从转染syk基因的Sf2 1细胞中提取、纯化免疫相关因子Syk蛋白。方法 :将syk基因转染Sf2 1细胞 ,于 2 8℃培养 48h ,收集细胞 ,用超声波破碎仪裂解细胞 ,提取裂解液中总蛋白 ,用Yellow 3凝胶和Toyopearl AF Heptin 650M凝胶层析柱分离、纯化。层析液中的Syk蛋白存在和性质 ,用SDS PAGE、免疫印迹实验和等电聚焦实验鉴定。结果 :从 2 5亿个Sf2 1细胞裂解液中提取了含有Syk的 2 2 5mg蛋白质。经Yellow 3凝胶层析分离 ,得到两个亚种的Syk蛋白 ,相对分子质量 (Mr)均为72× 10 3 。进一步用Toyopearl AF Heptin 650M凝胶层析纯化后 ,得到两个纯的Syk蛋白 ,SDS PAGE、免疫印迹实验结果显示 ,两种Syk的Mr 均为 72× 10 3,与Syk的理论相对分子质量吻合。但等电聚焦实验显示 ,这两种Syk蛋白成分具有不同的pI值。结论 :从 2 5亿个转染syk基因的Sf2 1细胞中纯化出8mgSyk蛋白 ,纯度高于 95%。这两种Syk的Mr 虽然相同 ,但具有不同的pI值 ,是两个亚种。这些Syk可用于研究Syk的作用机制、抗Syk抗体的制备和Syk诊断试剂盒的制备等     

Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

单克隆抗体2A7亲和层析法纯化红细胞膜表面的CD59分子

用抗ＣＤ５９单抗２Ａ７与ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ偶联制备亲和层析柱，从正常人红细胞膜蛋白抽提物中分离纯化出ＣＤ５９分子。本法纯化的ＣＤ５９分子量为１８－２０ＫＤａ对还原剂敏感，与ＭｃＡｂｓｓＭＥＭ４３和１Ｆ５有高亲和性，再掺入豚鼠红细胞后，能够抑制人体补体的反应性溶血，本文还对三株抗ＣＤ５９单抗２Ａ７，ＭＥＭ４３及１Ｆ５的抗原特异性进行了初步分析。     

Polyamines in Trichomonas vaginalis

Trichomonas vaginalis was grown in a modified Bushby's medium and putrescine, spermidine and spermine levels were determined in extracts from 24- and 48-h cultures and also in the culture media. All three polyamines were present in T. vaginalis extracts; the putrescine level and putrescine/spermidine ratio were much higher than those reported for other protozoa or for mammalian tissues. There were no significant differences between 24-h and 48-h amine levels per mg protein in these extracts, but amine levels per cell were higher at 24 than at 48 h. The spent culture media had a much higher putrescine content than corresponding uninoculated media and it was concluded that T. vaginalis secreted putrescine into the culture medium.     

Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort

Familial hypercholesterolaemia (FH) is a common inherited disorder, associated with premature vascular disease. FH may be caused by many different mutations in the low density lipoprotein receptor (LDLR) gene, about 700 mutations have been described, most of which occur rarely and often only in single families. Although particular mutations are prevalent in certain ethnic groups, countries with heterogeneous population bases (such as NZ) may carry a wide variety of mutations; making a gene screening approach the appropriate first step for a mutation detection programme. We have compared SSCP with DHPLC to assess their effectiveness as methods for LDLR mutation detection. Although five novel LDLR mutations were detected by SSCP in patients with FH, DHPLC was more sensitive, with eight novel mutations detected. Six of these mutations (T392M, R419G, Y421N, 1206-1207delCT, 1872delC, and 1943delC) were clustered in exons 9 and 13 of the EGF precursor homology domain, one (679-680delAC) in the ligand binding domain (exon 4) and the eighth (P774H) in the membrane-spanning domain (exon 16). Twenty five mutations were identified in 35 patients in total. Of these, we were able to detect only 64% of mutations by SSCP even though all variants were detected by DHPLC. All patients are heterozygous for the mutations, which is consistent with the clinical phenotypes.     

Tumor cell surface β1–6 branched oligosaccharides and lung metastasis

NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the1–6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in1–6 branched Asn-linked oligosaccharides.     

Physical separation of HLA-A alleles by denaturing high-performance liquid chromatography

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.