Western blot assay for prostate-specific membrane antigen in serum of prostate cancer patients

There is a need for the development of new diagnostic tools for the early detection of prostate cancer. A candidate molecule for a new screening test is a prostate-specific membrane antigen (PSM) recognized by the monoclonal antibody 7E11.C5. We carried out studies aimed at identifying PSM in the serum of normal and benign prostatic hyperplasia (BPH) donors and patients with adenocarcinoma of the prostate, in order to judge whether the development of a serum assay using this marker was feasible. By Western blotting, we found significant levels of PSM in serum samples from prostatic cancer patients, in the seminal fluid of pooled normal donors, in BPH patients, and in normal male sera. Similar to prostate-specific antigen (PSA), PSM was present in seminal plasma in higher concentrations than in serum, and PSM levels in prostatic cancer patients were significantly higher than in normal controls. These data suggest that the development of an assay utilizing the PSM and new monoclonal antibodies directed against the antigen, could provide a feasible test for prostatic cancers. © 1994 Wiley-Liss, Inc.     

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique.

The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.     

Purification of glial fibrillary acidic protein (GFAP) from normal bovine brain

Glial fibrillary acidic protein (GFAP) was purified from normal bovine brain by a modification of the procedure used to isolate vimentin in order to avoid contamination by other cytoskeletal components; vimentin, neurofilament triplet proteins, tubulin and actin. GFAP is thought to be separated from vimentin in the DE cellulose column chromatography step. The three other major proteins were also separable through ion exchange and gel filtration column chromatographies. A purified 49 kDa polypeptide was estimated to be GFAP from peptide mapping and subsequent immunoblotting analysis. We obtained 4.4 mg GFAP/1 g bovine brain white matter in less than 3 days. The polyclonal antibody raised against purified GFAP was able to detect 49 kDa GFAP by immunoblotting analysis. This isolation method is simpler and more rapid than previous methods.     

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-env gp46(188-224), as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, Cys-env gp46(188-224) of HTLV-I, is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46 (188-224) conjugate and Cys-env gp46 (188-224)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was more sensitive and useful than the immune complex transfer enzyme immunoassay using Cys-Arg-env gp46(188-209) and other methods using HTLV-I as antigen.     

姬鼠型流行性出血热鼠间传播因素的调查研究及多元线性回归分析

1989年12月,在江苏省姬鼠型疫区进行EHF鼠间传播因素调查,并用免疫转印技术对同一鼠窝中鼠螨分离的EHF病毒进行抗原多肽分子量测定,结果一致。以每窝鼠数、鼠皮肤破损数、鼠窝残留食物EHF抗原阳性数和革螨数为自变量,以鼠窝鼠EHF抗原、抗体阳性率和EHF总感染率为因变量,分别进行多元线性回归分析。结果:4个自变量和3个因变量的回归关系有非常显著性意义;进一步进行回归方程显著性检验表明,在姬鼠型疫区鼠间传播因素中,以皮肤破损为主要因素;鼠间密切接触和革螨叮咬为次要因素:而与食入EHF抗原污染的食物关系不密切。     

An improved procedure for the ''dot immunobinding'' analysis of hybridoma supernatants

A computer-assisted method for the positive/negative discrimination of ELISA data is described. This method was applied to a rapid ELISA procedure for IgG class antibodies to Aspergillus fumigatus in which the performance time of the test was reduced to 1 h. The method gives results which compare well with those by agar gel double diffusion (AGDD). The computer-assisted reading, calculation and tabulation of ELISA results from one microtitre plate is performed in less than 2 min and permits analysis of large numbers of specimens.     

Induction and activation of protein kinase C delta in hippocampus and cortex after kainic acid treatment

Various isoforms of protein kinase C (PKC), especially the novel PKC subtypes delta, epsilon, and the atypical subtype PKC zeta, are involved in delayed cell death. We studied the expression and late activation of the latter PKC isoforms in comparison with classic PKC alpha, beta, and gamma in the brains of rats exposed to systemic kainate injection. The expression of PKC delta mRNA was strikingly upregulated (13-fold) in the cortex and the CA1 and CA3 hippocampal regions on 1 day after kainate administration, whereas PKC zeta mRNA was only moderately increased (about 100%) in these three brain regions on day 2 following the drug. PKC epsilon mRNA was slightly increased only in the cortex on days 2 and 6, while the mRNA levels of the classic PKC subtypes (alpha, beta, and gamma) remained unchanged or decreased after the treatment. Immunoblotting analyses revealed that the level of PKC delta protein started to increase on day 1 after kainate and was significantly elevated on day 2 in both the membrane and cytosol fractions of cortex and hippocampus. PKC epsilon protein only showed a marginal increase and the level of PKC zeta protein remained unaltered in response to the treatment. Cortical and CA1-3 pyramidal neurons displayed strong immunoreactivity for PKC delta on days 1 and 2, and microglia on days 1, 2, and 4 after the drug. The results indicate that the expression of apoptosis-associated isoforms of PKC, most notably that of delta, but to lesser extent also that of epsilon and zeta, is increased during kainate-induced neuronal death. The predominant induction of PKC delta in neurons and microglia suggests that PKC delta could be the major mediator or modulator of apoptotic and inflammatory responses to excitotoxic insults.     

Monosialoganglioside protected ischemic rat hippocampal slices through stabilizing expression of N-methyl-D-aspartate receptor subunit

INTRODUCTION Monosialoganglioside(GM1) can alleviatecerebraledema and reduce cerebral infarct volume in in vivoanimal models[1-3], and can prevent death of culturedgranule cells in vitro from anoxia as well[4]. However,meaning of data derived from a neural preparation with-out a vasculature and mammalian systemicinteractionis limited. The direct protective effect of GM1 on is-subunits are mainly distributed in the forebrain[5], and μmol/L (control group), 0.01 μmol/L(gr…     

TrkA and mitogen-activated protein kinase phosphorylation are enhanced in sympathetic neurons lacking functional p75 neurotrophin receptor expression

This study examined the effects of hypomorphic p75 neurotrophin receptor (p75NTR) expression and high levels of nerve growth factor (NGF) on trkA phosphorylation and downstream activation of p44/42 mitogen-activated protein kinase (MAPK). Post-ganglionic sympathetic neurons from postnatal day 1 p75NTR exon III null mutant (p75(-/-)) and 129/SvJ mice were cultured in the presence of 50 ng/mL NGF and analysed by Western blotting. Levels of phosphorylated trkA are increased in p75(-/-) neurons compared with 129/SvJ neurons, and these higher levels are maintained with continuous exposure to NGF. MAPK is also phosphorylated to a greater extent in p75(-/-) neurons than in 129/SvJ neurons, both within 10 min of exposure to NGF, and with continuous NGF treatment for 5 days. These data provide new insight into the mechanism underlying enhanced neurite outgrowth in p75(-/-) neurons, demonstrating that trkA and MAPK signalling in sympathetic neurons are increased when p75NTR function is disrupted.