Cat allergen 1: Biochemical,antigenic, and allergenic properties

Cat allergen 1, an important agent in human allergic reactions, has been partially purified by affinity chromatography. Heating the purified allergen at 100 ° C for 30 min resulted in a 28% loss in the antigenicity of the allergen molecule (determined by Laurell rocket assay), although lower temperatures had little effect. Its allergenicity (determined by passive transfer skin test) was diminished slightly after heating to 56 ° or 100 ° C. Reduction with dithiothreitol or 2-mercaptoethanol resulted in greater losses of antigenicity and allergenicity but did not obliterate these properties. Three forms of the affinity-purified allergen (isoallergens) differing slightly in isoelectric point were demonstrated by isoelectric focusing followed by crossed immunoelectrophoresis. The molecular weight of cat allergen 1 under physiologic conditions was 35,000 ± 2000 as determined by gel filtration in Sephadex G-75. Under the dissociative conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with or without prior reduction by dithiothreitol, cat allergen 1 appeared to exist as an antigenically active subunit with a molecular weight of 18,000 ± 2000. This subunit molecular weight estimate was confirmed by gel filtration in 6M guanidine hydrochloride. The stability of the allergenic and antigenic activity of cat allergen 1 suggests that this activity may be determined partially by the primary sequence of allergenic sites on the molecule. The separation ond purification of molecular subunits may allow sequence analysis of these sites.     

In vitro differentiation of Trypanosoma cruzi under chemically defined conditions

Metacyclic trypomastigotes of Trypanosoma cruzi have been obtained in chemically defined axenic culture. The differentiating medium, composed of artificial triatomine urine supplemented with proline, allows high yields of metacyclic trypomastigotes after 72-h incubation of T. cruzi cells at 27 degrees C. Morphological differentiation of the parasites is gradual under these chemically defined conditions and is preceded by the expression of stage-specific polypeptides. The yield of in vitro-induced metacyclic trypomastigotes depends upon the age of the epimastigote culture, the size of the inoculum and the depth of the medium. Metacyclic trypomastigotes differentiated in vitro from the Dm 28c clone of T. cruzi are both resistant to complement lysis and to macrophage digestion. They are able to infect mice with an efficiency similar to that obtained for natural metacyclic trypomastigotes obtained from triatomine excreta.     

Effects of a 4-deoxyphorbol triester from the latex sap of Euphorbia biglandulosa Desf. on isolated spinach chloroplasts

The effects of a 4-deoxyphorbol triester (4-DPT) extracted from the latex sap of Euphorbia biglandulosa Desf. on isolated intact chloroplasts and thylakoid membranes from spinach leaves (Spinacia oleracea L. cvs. Monatol) have been investigated. Light-induced electron flow through photosystem II was highly sensitive towards the diterpene ester: half-maximal inhibition of ferricyanide reduction was obtained with 4 to 7 × 10^?7 moles 4-DPT/mg chlorophyll. In contrast, the ADP/2e^- ratio of noncyclic photophosphorylation which accompanies K₃[Fe(CN)₆] reduction remained nearly unaffected. At concentrations of 4-DPT which caused half-maximal inhibition of ferricyanide reduction, neither drastic changes in photosystem I-dependent photochemical reactions nor alterations in the permeability characteristics of the chloroplast envelope were observed. However, 4-DPT to some extent affects the permeability of the thylakoid membranes. From the data it can be concluded that 4-DPT, which has a strong inhibitory activity on rat liver mitochondria, also acts as a highly effective inhibitor of photosynthetic electron transport.     

Lens membranes. IV. Preparative isolation and characterization of membranes and various membrane proteins from calf lens.

A scheme is proposed for the preparative isolation of matrix-bound and matrix-free calf lens membranes, and the conditions and recoveries of the procedures have been studied. Gas chromatographic analyses have been carried out to characterize the sugars of the intrinsic glycoproteins. Galactose and N-acetyl-glucosamine are dominant sugar components and fucose, glucose and N-acetyl-galactosamine are minor constituents.Urea-treated membranes, prepared from cortex plus nucleus, contain only very small amounts of polypeptides which immunologically cross-react with α- and γ-crystallin and which can probably be classified as remnants. The weight ratio protein : cholesterol : phospholipid of these membranes amounted to 4·4 : 1 : 1·9, and the molar ratio cholesterol : phospholipid was 1·0, which is equal to those of the original lens tissue.A water-extractable protein fraction can be recovered from urea-treated membranes, which gives six bands in SDS-gel electrophoresis (mol. wt. 54 000, 47 000, 34 000, 27 000, 19 000 and 16 000). This fraction contains some α- and γ-crystallin according to immunological and electrophoretical criteria. Its major component has a molecular weight of 47 000.     

In vitro induction of apoptosis vs. necrosis by widely used preservatives: 2-phenoxyethanol, a mixture of isothiazolinones, imidazolidinyl urea and 1,2-pentanediol

Preservatives are added to many final products, such as detergents, cosmetics, pharmaceuticals and vaccines. We conducted an in vitro investigation of the apoptosis- and necrosis-inducing potential of brief applications (10 min) of four common preservatives: ethylene glycol monophenyl ether, 2-phenoxyethanol (EGPE), imidazolidinyl urea (IMU), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMI/MI), and 1,2-pentanediol, a "preservative-non-preservative" best known as pentylene glycol. Using HL60 cells, we monitored the kinetics of cell toxicity with the MTT test and analysed extranuclear end points of apoptosis, i.e. phosphatidylserine exposure and nuclear fragmentation. Preservative treatment resulted in a dose-dependent decrease of cell viability. The mode of cell death was dose-dependent: necrosis occurred at high concentrations while apoptosis, shown by DNA laddering, DNA sub-diploid peak and caspase-3 activation, occurred at lower concentrations 0-24hr after exposure to a single dose: CMI/MI induced apoptosis at low concentrations (0.001-0.01%) and necrosis at high concentrations (0.5-0.1%); IMU and EGPE required higher concentrations to induce apoptosis (IMU 0.01-0.1% and EGPE 0.01-0.5%) or necrosis (IMU 0.5-1% and EGPE only at 1%). PG induced apoptosis only at 5%. Externalization of PS, a hallmark of apoptosis, occurred early in HL60 treated with low concentrations of CMI/MI and EGPE and was concomitant with the subdiploid peak in HL60 treated with PG. However, it did not occur in HL60 treated with IMU. In conclusion, at appropriate concentrations, each of the four preservatives modulates the apoptotic machinery by a caspase-dependent mechanism. Thus, apoptosis could be a good parameter to evaluate the cytoxicity of these chemical compounds.     

Mechanism of action of non-cisplatin type DNA-targeted platinum anticancer agents: DNA interactions of novel acridinylthioureas and their platinum conjugates

The DNA binding of two novel acridinylthioureas, ACR-NH-(CH(2))(2)-C(S)-NHCH(3) (1) and ACR-N(CH(3))-C(S)-NHCH(3) (3), and their platinum conjugates 4 and 5-derived from [PtCl(2)(en)]-was studied in cell-free model systems using various physico-chemical and biophysical methods. These included: spectrophotometric drug-DNA titrations, ethidium-DNA fluorescence quenching, competitive drug displacement, high-resolution NMR spectroscopy, and unwinding of plasmid DNA monitored by agarose gel electrophoresis. The acridinium cation of 1 showed strong binding to native DNA with K(i)=1.5 x 10(6)M(-1) and an excluded site size (n) of 2bp (McGhee-von Hippel fits of absorbance data). Compound 3 showed no measurable association with DNA. Binding of 1 was an order of magnitude stronger than that of simple 9-methylaminoacridine (2). In alternating copolymers, 1 exhibited slight AT preference. In poly(dA-dT)(2), enhanced association was accompanied by an increased binding site (approximately 3bp), while parameters in poly(dG-dC)(2) were consistent with classical intercalation. Displacement of 1 by distamycin from calf thymus DNA was suggestive of non-intercalating thiourea in 1 being located in the minor groove of the duplex. 1H NMR data of d(GGAGCTCC)(2) modified with 1 indicated intercalative binding of planar acridine, based on upfield shifts of aromatic proton signals relative to those in unbound 1 (Deltadelta approximately equal to -0.5 to -1ppm). Finally, 4 and 5 were found to unwind negatively supercoiled pUC19 plasmid by 21 degrees and 7 degrees per adduct, respectively (electrophoretic gel mobility assays). The difference in DNA binding modes of 4 and 5 is discussed as the ultimate source of the distinctly different biological activities of the conjugates.     

Homogeneous colorimetric enzyme inhibition immunoassay for cortisol in human serum with Fab anti-glucose 6-phosphate dehydrogenase as a label modulator

The Fab fragment of rabbit IgG antibody to bacterial glucose 6-phosphate dehydrogenase covalently linked to the cortisol retained the capacity to inhibit the enzyme completely. In optimal conditions the antibody to cortisol effectively bound the cortisol residues of the cortisol-Fab conjugate, making it incapable of inhibiting the enzyme. The enzyme modulatory properties of the cortisol-Fab conjugate were exploited to set up a direct competitive homogeneous enzyme immunoassay for cortisol in human serum. The procedure involved the use of the auxiliary enzyme diaphorase, specific for NADH, which converts the nitro blue tetrazolium salt to a colored formazan. The procedure detects modulated glucose 6-phosphate dehydrogenase activity by a single-point measurement without serum interference. The assay working range was between 20 and 640 micrograms/1 of cortisol and used 50 microliters of sample.     

小花八角枝叶中的薄荷烷型单萜

目的研究有毒药用植物小花八角Illicium micranthum枝叶的化学成分及其对韭菜迟眼蕈蚊幼虫的毒杀活性。方法利用硅胶柱色谱、Sephadex LH-20凝胶柱色谱等方法对小花八角枝叶的化学成分进行系统的分离,通过质谱、核磁共振谱等波谱方法,结合理化性质分析鉴定化合物结构;采用触杀胃毒联合毒力方法测定化合物2对韭菜迟眼蕈蚊幼虫的毒杀活性。结果从小花八角枝叶甲醇提取物的醋酸乙酯萃取部位分离得到5个薄荷烷型单萜化合物,分别鉴定为(-)-(1R*,4S*)-1-羟甲基-2-烯基-4-异丙基-1,4-环己二醇(1)、(1R,2R,4R)-1-甲基-4-异丙基-1,2,4-环己三醇(2)、4-羟基胡椒酮(3)、乳香醇C(4)、α,α-二甲基-4-羟甲基苯甲醇(5)。其中化合物1为新化合物,命名为小花八角素A。结论化合物1为新化合物,化合物2~5为首次从该植物中分离得到。化合物2对韭菜迟眼蕈蚊幼虫的毒杀活性与浓度呈正相关,48 h后的LD50值为30.4 mg/L,72 h后的LD50值为22.5 mg/L。     

苯基苯酚型酚醛树脂中羟甲基邻/对位异构的核磁共振分析及性能影响

采用核磁共振光谱(NMR)技术对苯基苯酚型酚醛树脂进行了结构分析.通过1H-NMR和13C-NMR计算树脂分子结构中邻、对位羟甲基相对含量比,发现苯基苯酚型酚醛树脂的相对含量比(5.0:1～4.3:1),远大于一般氨酚醛树脂(1.9:1～1.2:1).同时对几种酚醛树脂的热性能、残碳率、贮存稳定性以及工艺温度下粘度的变化特性进行了比较研究,从微观角度为苯基苯酚型酚醛树脂具有各种优良性能找到了理论依据.     

Conventional and novel PET tracers for imaging in oncology in the era of molecular therapy

In the last ten years, the development of several novel targeted drugs and the refinement of state of the art technologies such as the genomics and proteomics and their introduction to clinical practice have revolutionized the management of patients affected by cancer. However, everyday practice points out several clinical questions: the difficulty of response assessment to new drugs especially using standard RECIST criteria that do not provide information on biological, vascular or metabolic variations; the inadequate selection of patients who are likely to benefit from a targeted therapy excluding those with breast cancer and gastrointestinal stromal tumours; the need to know the global biological background of diseases especially in metastatic setting using repeatable non-invasive procedures. Molecular imaging could provide information on in vivo distribution of biological markers in response to targeted therapy and could improve the selection of patients before therapies. The aim of this review is to analyze the current role of conventional and innovative positron emission tomography (PET) radiotracers in clinical practice and to explore the promising perspectives of molecular imaging in cancer research.