用DNAG C含量和DNA同源性测定法鉴定米克戴德军团菌

本文采用热变性温度法和液相复性速率法对—轻型特征及血清学反应相似米克戴德军团菌(Lm)的菌株进行了测定,结果表明该菌与标准Lm(C DC株)的DNA G Cmol％相差3.45％,与标准Lm(C DC株)的DNA同源性达81.99％,根据伯杰细菌鉴定手册(1984),可判定该菌株与标准Lm(C DC株)为遗传型一致的类群,即从遗传学角度证明该菌为Lm。     

Cyrene™ as an Alternative Sustainable Solvent for the Preparation of Poly(lactic-co-glycolic acid) Nanoparticles

Toxic and environmental harmful organic solvents are widely applied to prepare poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) in standard preparation methods. Alternative non-toxic solvents suffer from disadvantages like high viscosity and plasticizing effects. To overcome these hurdles, Cyrene? as a new sustainable, non-toxic and low viscous solvent was used to formulate PLGA NPs. A new preparation method was developed and optimized. Small sized blank NPs around 220 nm with a narrow size distribution and highly negative charge (<?23 mV) were obtained. To test the application for drug delivery, the lipophilic model drug atorvastatin was encapsulated in high drug loads with comparable physicochemical characteristics as the blank NPs, and a total drug release within 24 h. No changes of the crystallinity or plasticizing effects could be observed. Highly purified NPs were obtained with a residual Cyrene? content <2.5%. Finally, the biocompatibility of Cyrene? itself and of the NPs formed in the presence of Cyrene? was demonstrated in a hen's egg test. Conclusively, the use of Cyrene? as solvent offers a simple, fast and non-toxic procedure for preparation of PLGA NPs as drug delivery systems circumventing the downsides of standard methods.     

Reduced surface area in apoptotic rounding of human chang liver cells from serum deprivation

Background: The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and “condensation of the cytoplasm” in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding. Methods: Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied (1) the cell cycle from propidium iodide–DNA bindings, (2) uptake of neutral red (NR) dye, a viable cell marker, and (3) cytosolic pH (pH_i) modulations from 2′,7′-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH₄Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy. Results: Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary A_o (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H⁺]_i/[H⁺]_o gradient promoting proton extrusion. The pH_i upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pH_i upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells. Conclusion: Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pH_i upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pH_i upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death. © 1994 Wiley-Liss, Inc.     

Immunopurification of human plasma kininogens

Human plasma kininogens were purified by immunoadsorption on Sepharose columns using two different approaches, either removing protein impurities with the respective immunospecific polymers or applying an anti-kininogen-specific immunoadsorbent column. An anti-kininogen serum developed and investigated in this laboratory in earlier studies was used. This antiserum recognizes the native conformational determinants in the kininogen heavy chain, the common denominator in plasma kininogens, and reacts with three heterogeneous molecular forms of high mol. wt kininogen (mol. wts 103,000, 92,000 and 90,000) as well as with low mol. wt kininogen. Heterogeneity of kininogens was shown by SDS gel electrophoresis and immunoelectrophoresis. With the antibody-specific polymers the yield was 80-100% compared to 75% or lower when several consecutive immunoadsorption steps were applied to remove impurities. Both methods serve the purpose of preparing immunologically pure kininogens suitable for immunization.     

Stimulation of haemopoietic stem cell proliferation: Characteristics of the stimulator-producing cells

Media conditioned by regenerating murine bone marrow cells contain a stimulator of haemopoietic stem cell proliferation. Fractionated cell populations have been examined for production of this stimulatory activity in order to characterize its cellular source. The stimulator is produced by adherent, phagocytic radioresistant, Thy 1.2^?, Fc⁺ cells in a population concentrated in a density range of 1.064 – 1.072 g/ml. The results indicate that the producer cells reside in the heterogenous mononuclear phagocytic population of the bone marrow.     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.     

Complexity and content of the DNA and RNA in Trypanosoma cruzi

The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 x 10(8) nucleotide pairs (2.8 x 10(-13) g) and a kinetoplast DNA content of 4.9 x 10(7) nucleotide pairs (5.4 x 10(-14) g). Reassociation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 x 10(3) present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments. By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A)+-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNA's of different abundancy levels: two sequences which occur ca. 3000 per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1-2 times per cell.     

HCG磁性微粒分离免疫酶联检测法及临床意义

为了观察正常妊娠妇女、宫外孕及葡萄胎患者血清中ＨＣＧ含量变化规律,该实验室启用一种新方法 ──磁性微粒分离免疫酶联检测法测定血清中ＨＣＧ浓度。该法灵敏度为１ｍＵｍｌ,精密度（ｘ±ｓ）为２３．３±０．５５、１２３± ２．１、３３６±１０．９,批内变异系数分别为２．４％、１．７％、３．２％。通过对正常妊娠不同孕期的妇女血清ＨＣＧ浓度测定,整个 妊娠期血清ＨＣＧ浓度变化呈双峰曲线,孕期在 ７０ ｄ左右时有一高峰,浓度达到 １００ ０００ ｍＵ／ｍｌ,此后呈下降趋势,在 ２６０ ｄ左右又有一小峰,浓度为 ２２ ０００ ｍＵ／ｍｌ,随后稍有下降直至分娩。 １２例宫外孕患者中,血清ＨＣＧ浓度除 １例外均 低于正常曲线。２例葡萄胎患者血清ＨＣＧ浓度远远高于正常曲线。此法测定ＨＣＧ浓度可更早地诊断早孕,还可以作为 宫外孕、葡萄胎的早期辅助诊断。磁性微粒分离免疫酶联法测定血清ＨＣＧ浓度其灵敏度、特异性已在临床实验中得到验 证,该法简便、快捷,没有放射性污染,实验产物无毒易处理,实用于临床。     

Analysis of structural differences between Ly-5 molecules of T- and B-cells

A group of closely related high mol. wt (Mr) membrane glycoproteins is expressed with varying Mr on different subpopulations of lymphocytes, but the different Mr forms share the Ly-5 and T200 antigenic determinants. The Ly-5 molecule expressed by thymocytes has an Mr of 175,000 (175ly5). The antigenically related molecule on B-cells has an Mr of 210,000 (210ly5). It is not known whether the variations in size are due to differences in the polypeptide chain, post-translational modifications such as glycosylation, or both. In this report we examine the glycosylation of 175ly5 and 210ly5 to determine whether differences in carbohydrate moieties may account for the different Mr of these two Ly-5 species. Pronase digestion and alkaline borohydride treatment of these molecules labeled in the terminal galactose residues revealed that 210ly5 molecules have a more complex oligosaccharide pattern than 175ly5 molecules. While Ly-5 oligosaccharides from a T-cell tumor line were very similar to those of normal thymocytes, the pattern of Ly-5 carbohydrates from a B-cell tumor were somewhat different than those from normal B-cells. This report also presents evidence for O-linked sugars on Ly-5 molecules.