Different calcium phosphate bioglass ceramics implanted in rabbit cortical bone. An interface study

In order to study the interface of calcium phosphate bioglass ceramics, cylinders of standard size were implanted in the tibiae of rabbits. The materials were evaluated by radiography, light microscopy and microradiography. Bioceramics with hydroxyapatite surface give rise to a closer contact with new bone than calcium phosphate glass ceramics.     

Immunofluorescent detection of anti-cytoskeleton antibodies using vinblastine-treated mononuclear cells

A reliable and reproducible immunofluorescence method is described for the detection of anti-cytoskeleton antibodies in human sera, based on the use of vinblastine-treated peripheral blood mononuclear cells as substrate. Three immunofluorescence patterns associated with antibodies to microfilaments, intermediate filaments and microtubules are readily identified.     

Localization of substance P- and enkephalin-like immunoreactivity in relation to catecholamine-containing cell bodies in the cat dorsolateral pontine tegmentum: an immunofluorescence study

The distribution of tyrosine hydroxylase-, substance P- and enkephalin-immunoreactive neurons in the cat dorsolateral pons was studied using the indirect immunofluorescence method of Coons. To allow for the visualization of substance P- and enkephalin-immunoreactive cell bodies, colchicine was injected either in the ventricular space or in the cerebral tissue. The distribution of the tyrosine hydroxylase-immunoreactive cell bodies corresponded with the well-known distribution of catecholamine cells in this area of the brain. The observation of adjacent sections treated separately with tyrosine hydroxylase- and enkephalin-antiserum revealed that most catecholaminergic cells contain enkephalin-immunoreactivity. In addition to this catecholamine-enkephalin cell population, a moderate number of substance P-immunoreactive cell bodies was found in dorsolateral pons. The peribrachial nuclei were found to be densely supplied with substance P- and enkephalin-immunoreactive fibers, whereas the medial subdivisions, which contain the majority of the catecholamine cells in the dorsolateral pons, display a moderate number of immunoreactive fibers. These results are suggestive of interactions between peptide-containing and catecholaminergic neurons and also between-peptide-containing and non-catecholamine-containing neurons in the cat dorsolateral pons.     

Mechanisms of the CYNAP phenomenon: evidence in the Bw49/Bw50 model for epitopes with different spatial orientation of antibody

The mechanism of the cytotoxic-negative, absorption-positive (CYNAP) phenomenon was studied using the model of the Bw49/Bw50 split of the BW21 antigen. Anti-Bw49 antibody bound 60% as well to Bw 50 as to Bw49 cells; however, even at a cytotoxic titer of 64 against Bw49 cells, the antibody was not cytotoxic to Bw50 Cells. At equal numbers of antibody molecules bound, the anti-Bw49 antibody activated C4 and C3, and induced lysis for Bw49 but not for the Bw50 cells. These data are consistent with a model in which different spatial orientations for shared epitopes can account for CYNAP reactivity for at least some selected Bw4/Bw6-associated splits of B locus antigens.     

Immunochemical characterization of mouse brain-associated theta (Thy-1) antigen.

The Thy-1 antigens or rat brain and thymus have been isolated and chemically characterized, but those of mice have not been identified. Moreover, it is uncertain whether the antigens are glycolipids or glycoproteins. This study with highly purified preparations of gangliosides GM₁, ¹GD_1a, GD_1b and GT_1b from bovine brain and several ganglioside fractions from mouse brain showed that Thy-1 activity does not reside in gangliosides, but rather in the chloroform-methanol-insoluble residue of brain remaining after extraction of gangliosides. The antigen could be solubilized from this residue with a non-ionic detergent. The antigenic activity of the solubilized preparation was heat-labile but resistant to periodate. The chemical properties of the Thy-1 antigen of mouse brain are discussed.     

用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应 (PCR)对蓝氏贾第鞭毛虫 (Giardialamblia)的磷酸丙糖异构酶 (triosephosphateisomerase ,缩写为tim)基因进行特异性扩增 ,结果扩增出 1条 6 83bp的DNA片段。此方法的特异性可高达10 0 % ,而其它DNA样本 ,如日本血吸虫 (Schistosomajaponicum)、刚地弓形虫 (Toxoplasmagondii)、微小隐孢子虫 (Cryptosporidiumparvum)、溶组织内阿米巴 (Entamoebahistolytica)、旋毛虫 (Trichinellaspiralis)和阴道毛滴虫 (Trichomonasvaginalis) ,以及人体血细胞等均未出现扩增反应。本法的敏感性也很高 ,可检测到0 4pg贾第虫包囊的DNA。 13株来自不同地理位置和 或宿主的贾第虫DNA样本在PCR中均各产生 1条长为 6 83bp的目的片段。上述结果表明本实验建立的检测贾第虫的PCR方法有效     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

 Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/P_i) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/P_i-cotransport, we measured type II Na/P_i-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10^–8 M) and/or vanadate (10^–3 M) for 3 h led to a non-additive 1.4-fold increase in Na/P_i-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/P_i-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/P_i-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/P_i-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/P_i-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/P_i-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/P_i-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules. Received: 1 May 1998 / Received after revision: 25 August 1998 / Accepted: 1 September 1998     

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.     

On the correlation between alkaline phosphatase and phosphate transport in rat renal brush border membrane vesicles

Alkaline phosphatase activity in renal cortex homogenates and in isolated brush border membrane vesicles was compared with the rates of sodium-dependent transport of inorganic phosphate (P_i) by isolated brush border membranes. Brush border membrane vesicles were isolated from renal cortical homogenates of rats adapted during a period of 5–7 weeks to diets with different dietary contents of P_i (low P_i diet=0.15 g%, high P_i diet=2.0 g%).Alkaline phosphatase activity was not increased in the low P_i diet group as compared to the standard P_i diet group but was reduced in the high P_i diet group. Sodium-dependent transport of P_i was increased 2–3-fold in the low P_i diet group as compared to the standard P_i diet group, whereas transport activity was only unsignificantly decreased in the high P_i diet group.Studying kinetik parameters in the two extreme dietary groups it has been found that these differences are due to alteredV _max of the transport activity as well as of alkaline phosphatase activity. TheK _m for both activities remained unaltered.Alkaline phosphatase activity and transport of P_i in brush border membrane vesicles were also compared in the presence of EDTA or Zn²⁺ at concentrations which inhibit alkaline phosphatase activity. Transport of P_i was not affected by the inhibitors even when alkaline phosphatase was inhibited by more than 70% (0.5 mmol/l Zn²⁺) or completely (0.5 mmol/l EDTA).The experiments suggest that no correlation between alkaline phosphatase activity and transport of P_i exists in isolated brush border membrane vesicles.