Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Localization of specific antigen in the organs of newborn animals vaccinated with liver smallpox vaccine]

Of 20 suckling rabbits, 4-5-days old, inoculated with live smallpox vaccine intradermally 6 displayed symptoms of generalized pox virus and neuroparalysis complications. Intensive accumulation of specific antigen in the brain, lungs, spleen, and the lymph glands was revealed by immunofluorescent method. The smallpox vaccine virus was isolated from these organs. Prolonged persistance of the attenuated smallpox virus was observed in the brain, spinal cord, lungs, spleen, and the lymph glands of 14 suckling rabbits showing no signs of any disease; specific antigen was revealed by immunofluorescent test. Vascular disturbances and slight cell changes were observed in the brain tissue of the inoculated animals. These changes were more severe in the sick animals.     

Comparative immunochemical and physicochemical study of the neurospecific antigen 10-40-4 from human and rat brains

Immunological non-identity of neurospecific protein 10-40-4 from various mammalian species has been demonstrated. Significant immunochemical differences were found between the protein 10-40-4 from the brain of rat, guinea pig and man. With respect to their physicochemical properties, no significant differences were found between individual neurospecific proteins 10-40-4 from the brain of man and rat. Therefore, immunological differences in the structure of a neurospecific determinant together with identical physicochemical properties of the proteins investigated imply that the latter are presented by species variations of the same protein.     

Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.     

Synthesis and in vivo evaluation of [18F]-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide as a PET imaging probe for COX-2 expression

Synthesis of [18F]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ([18F]celecoxib), a selective COX-2 inhibitor, is achieved via a bromide to [18F]F- exchange reaction. Synthesis of the precursor for radiolabeling was achieved from 4'-methylacetophenone in four steps with 22% overall yield. Under non-radioactive conditions, fluorination was achieved using TBAF in DMSO at 135 degrees C in 80% yield. Synthesis of [18F]celecoxib was achieved using [18F]TBAF in DMSO at 135 degrees C in 10+/-2% yield (EOS) with >99% chemical and radiochemical purities. The specific activity was 120+/-40 mCi/micromol (EOB). [18F]celecoxib was found to be stable in ethanol, however, de[18F]fluorination (6.5%) was observed after 4 h in 10% ethanol-saline solution. Rodent PET studies show bone labeling indicating in vivo de[18F]fluorination of [18F]celecoxib. PET studies in baboon indicated a lower rate of de[18F]fluorination than rat and retention of radioactivity in brain regions consistent with the known distribution of COX-2. A radiolabeling method that can generate consistent high specific activity is needed for routine human use.     

Use of the ELISA immunoenzyme method for the detection of the causative agent of tularemia

The conditions of the enzyme-linked immunosorbent assay (ELISA) for the detection of Francisella tularensis were worked out. In the study of 27 strains differing in their biological characteristics, the sensitivity of the assay was determined, varying within the range of 1 X 10(4)--5 X 10(4) million cells/ml and exceeding the sensitivity of the currently used methods for the immunodiagnosis of tularemia by 1-2 orders. ELISA also proved to be a highly effective technique for the detection of the specific antigen in the organs of infected animals. The antigen was regularly detected in the organs of white mice, beginning from day 3 after their infection with the minimal doses of F. tularensis. The method may be recommended both for the identification of isolated cultures and for the early diagnosis of tularemia infection.     

Identification, purification and physico-chemical properties of neurospecific alpha 1-globulin]

The partially purified and concentrated 500-600-folds protein fraction has been obtained from human brain extract. This protein fraction was used for the immunization of rabbits. The corresponding anti-sera have the potency to detect the brain specific alpha 1-globulin, which is not identical to known cytoplasmatic brain specific protein. These antisera were used for the control of the antigen purification procedure which included ion-exchange, affinity and hydropho'ic chromatography, gel-filtration and isochromatofocusing. The antigen, purified to the homogeneity, has the electrophoretic mobility of the alpha 1-globulins, M(r) = 110-10 kD, and isoelectric point at pH 2.9-3.1.     

Identification, isolation and physico-chemical properties of neurospecific alpha2-globulin]

In the immunization process of rabbits with the protein fraction of the water-salt extract from human brain partially and concentrated at 500-600 times was received antiserum revealed brain specific alpha 2-globulin that is not identical to the known cytoplasmatic brain specific antigens. This antigen has got electrophoretic mobility of alpha 2-globulins, molecular weight 90 +/- 10 kD and isoelectric point 4.1-5.4. Develop the procedure for purification of this antigen on the basis of the combination ion change, affinity, hydrophobic chromatography gel-filtration and isochromatofocusing.     

Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments.

A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.     

黄鳝IRF-4和IRF-10的表达研究

目的:干扰素调节因子是一类能够调控干扰素及其相关免疫基因表达的转录因子,研究黄鳝干扰素调节因子的结构及表达有助于阐明黄鳝抗病毒的机理。方法:利用PCR扩增技术获得了黄鳝干扰素调节因子10(IRF-10)和IRF-4的部分cDNA序列,再利用半定量PCR技术检测了黄鳝不同发育阶段、不同组织IRF-10和IRF-4的表达。结果:IRF-10和IRF-4在黄鳝三个不同的发育阶段表达量基本一致,但两者在黄鳝不同组织表达呈现明显的差异,IRF-10组成型表达于黄鳝各个组织中,而IRF-4仅在肠、中肾和脑中呈现很高的表达,其他组织表达很弱。结论:IRF-10组成型地表达于黄鳝各个组织,且表达量很高;而IRF-4中仅在主要免疫器官表达,且表达量较弱。     

Cellular and regional localization of the neurospecific antigen 10-40-4 in the human brain

Cellular and regional localization of neurospecific protein 10-40-4 in human brain was studied by immunofluorescent staining and immunoenzymatic assay. Intense fluorescence of perikaryons of the medulla oblongata, thalamus and pons neurons was demonstrated. The same structures showed the maximal concentration of the protein (15-18% of water-soluble proteins). In the cortex of the hemispheres, in the cerebellum and hypothalamus the fluorescence intensity was not different from the background level. The concentration of the protein in these structures was minimal (1-4% of water-soluble proteins).     

Effect of immunization with neurospecific proteins and tubulin on learning in the rat

The paper deals with the effect of anticerebral antibodies on rats learning in T-shaped maze with food reinforcement. The rats of August line were immunized by neurospecific proteins 10-40-4 from the human and rat's brain, 14-3-2 and cerebral tubuline. Rats immunized by bovine serum albumine (BSA) served as a control of the influence of antibodies to noncerebral protein. Rats injected with Freund adjuvant and saline, served as control of Freund adjuvant action. At the time of antibodies formation (from the 7-th day after the last injection) the rats were trained in a T-shaped maze during 4 days. The acquisition of conditioned reactions was inhibited in rats immunized with neurospecific proteins 10-40-4 from the rats brain, 14-3-2 and cerebral tubuline. Antibodies to neurospecific protein 10-40-4 from the rats brain produced the greatest effect on the process of learning. They elicited a significant decrease of conditioning and an increase of the number of errors. Antibodies to the neurospecific protein 10-40-4 from the human brain and to the BSA, i.e. to proteins which are not present in the nervous tissue of the rats, did not affect the learning. The obtained results indirectly confirm the permeability for the antibodies of the blood-brain barrier in immunized animals.     

Comparison of cytoplasmic superoxide dismutase in liver,heart and brain of aging rats and mice

Comparisons of the specific activity of cytoplasmic superoxide dismutase, in homogenates of liver, brain and heart demonstrate considerably reduced activity in livers of aging rats and mice, a very small reduction in specific activity in the heart and no reduction in the brain of aging animals.Antisera elicited in rabbits against purified superoxide dismutase from liver of either young or old rats revealed complete cross-reactivity with the heart and brain enzymes. They also exhibited complete cross-reactivity with the mouse enzymes from all three organs. This finding has, for the first time, enabled a comparison of possible age-dependent alterations in the same enzyme antigen in different cell types.Despite the differences in age-related changes in specific activity in homogenates of liver, heart and brain the enzyme shows a considerable decline in catalytic activity per antigenic unit in $\text{all}$ three organs in both aging rats and mice.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.     

An anti-probasin monoclonal antibody recognizes a novel 40-kDa protein localized in rat liver and a specific region of kidney urinary tubule.

Immunoblotting with a monoclonal antibody against probasin (rat prostatic secretory protein) showed that a 40-kDa protein antigenically related to probasin was localized in rat liver and kidney. The contents of probasin in these organs were negligible. Immunostaining revealed that the 40-kDa protein (probasin-related antigen: PRB-RA) was expressed in the liver parenchymal cells and the kidney urinary tubular epithelial cells in outer stripe. The content of PRB-RA in the kidney was low during 0 to 2 weeks of age, then rapidly increased about 10-fold from 2 to 8 weeks of age. The content in the liver increased about 2-fold during the period, reaching a value of 10-12 ng/micrograms protein, which was ten times higher than that in the kidney. PRB-RA was purified from rat liver by ion-exchange chromatography, gel filtration and fast protein liquid chromatography on a hydroxyapatite column. The purified protein formed insoluble aggregates in the absence of a detergent, and it had a blocked amino terminal. The amino acid sequence of a peptide generated by tryptic digestion of alkylated PRB-RA was determined. Computer analysis showed that there was no protein having a significant homology with the peptide. These results indicate that a novel 40-kDa protein with a structural similarity to probasin is localized in rat liver and kidney, and might bear a function specific to these organs.     

THE NATURE OF THE TWO PROTEINS OF BRAIN SPECIFIC ANTIGEN 14-3-2

The three isoenzymes of rat brain enolase (2-phospho d -glycerate hydrolase EC 4.2.1.11.) χχ, χγ and γγ were separated by ion-exchange chromatography and were tested for reaction with an antiserum against brain specific antigen 14-3-2. This monospecific antiserum affects the enolase activity of only the χγ and γγ isoenzymes. Immunoelectrophoretic experiments show that the two proteins which react as 14-3-2 both contain γ enolase subunits, and one of these also contains χ enolase subunits. It is concluded that the 14-3-2 antigen and the γ enolase subunit are identical, and that the two proteins which react immunologically as 14-3-2 are the χγ and γγ enolase isoenzymes.     

Effect of RO 15-1788 and RO 5-4684 on the evoked activity of neurons in hippocampal sections

The action of specific benzodiazepine (BD) antagonist R015-1788 and peripheral benzodiazepine receptor (BDR) ligand R05-4864 on the evoked activity of hippocampal neurons was studied using brain slice method. The extracellular activity was registered in CAI area upon single and paired pulse stimulation of Schaffer collaterals. R015-1788 application (5 microM, for 3-6 min) reduced paired pulse inhibition (PPI). More prolonged application produced a depression of the population spike (PS). R015-1788 (5 microM) blocked diazepam (2 microM), hexobarbital (10 microM) and GABA (40 microM) potentiation of PPI. Interaction of R015-1788 with endogenous BD-like ligand as a possible explanation for the effects under study is discussed. R05-4864 (10 microM) reduced PPI and decreased PS evoked by single pulse stimulation. Frequency stimulation revealed the generation of additional PS after drug application. The data presented suggest that suppression of hippocampal inhibitory circuits may be a general feature of anxiogenic BDR ligands.     

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.