一种快速分离细胞凋亡片段化DNA的简单方法--NP-40法

目的 鉴定细胞凋亡的一个重要生化指标是凋亡细胞的核ＤＮＡ通过琼脂糖凝胶电泳呈现典现的ＤＮＡ“梯状”图谱。传统分离凋亡细胞片段化ＤＮＡ的方法多采用酚／氯仿抽提。因此,有必要探寻一种更为灵敏、简单的分离凋亡片段化ＤＮＡ的新方法。方法 通过比较传统分离凋亡片段化ＤＮＡ的方法,并对ＨＥＲＲＭＡＮＮ等报告的方法加以改进。结果 本文介绍一种快速分离凋亡ＤＮＡ片段化的新方法（ＮＰ－４０法）。结论 这种简单、快速     

A simple method for the isolation of murine peripheral blood lymphocytes

A simple, rapid and reproducible semi-microtechnique was developed for the isolation of murine peripheral blood lymphocytes. Murine peripheral blood was collected from brachial vessels and centrifuged through a Ficoll--Hypaque gradient in a microfuge tube yielding 1--1.5 X 10(6) lymphocytes per mouse.     

A simple method for assessment of glomerular size and its use in the study of kidneys in acromegaly and compensatory renal enlargement

As measurement of absolute glomerular size is difficult we developed a method of assessing glomerular size that was simple and practical and could be used to compare the kidneys in different groups of patients. Using a semi-automatic image analyser, the cross-sectional area of 100 randomly-selected glomeruli, outlined by Bowman's capsule, was measured on sections of kidneys taken at necropsy. The mean of the logarithms of the largest 25 areas was calculated. The method was applied to compare control kidneys (53) with the kidneys in acromegalics (20), in patients with one kidney (10) and in patients with asymmetrical kidneys (12). Kidneys were heavier in the three test groups than in controls. Glomerular sizes were similar in controls and in acromegalics but were larger in single and disparate kidneys. There was a relationship between glomerular size and kidney weight within the control group and across the four groups taken together. This only partly accounted for the observed differences in glomerular size between the groups. Histological comparison of the acromegalic and single kidneys showed more global glomerulosclerosis in single kidneys and also segmental lesions, mainly at the glomerular hilum, only in the single kidneys. These findings show that renal enlargement occurs in acromegaly and in single and disparate kidneys but is accompanied by markedly different glomerular features. This implies different mechanisms for the renal enlargement. The method of assessing glomerular size is useful in the study of these and other conditions affecting the kidney.     

一种分离新生小鼠肝细胞的简单方法

目的建立新生小鼠肝细胞体外分离培养方法。方法采用胶原酶消化组织块法分离新生小鼠肝细胞，倒置显微镜动态观察细胞形态特征，并进行鉴定：PAS（periodieacid-Schiff，PAS）染色观察细胞肝糖元的含量；免疫组化SP法检测细胞甲胎蛋白AFP（alpha fetal protein，AFP）的表达；生化法检测肝细胞培养上清白蛋白的含量。结果肝组织块经用胶原酶直接消化，获得的肝细胞成活率达80％以上。肝细胞培养一周后长满瓶底，呈集落生长，融合成片。培养的肝细胞PAS糖原染色阳性；AFP表达阳性；培养上清白蛋白含量至培养5～7d时迭峰值，随后又逐渐下降。结论胶原酶消化组织块法分离新生小鼠肝细胞，方法简单、高效，培养的原代肝细胞符合新生肝细胞的生物学特性。     

A simple method of chromosomal analysis for malignant solid tumors.

A case of essential thrombocythemia with a partial deletion of the long arm of chromosome 11, del(11)(q21) as a sole chromosomal anomaly is reported. Rearrangement of chromosome 11 at band 11q21 has been reported in six patients with chronic myeloproliferative disorders: four with post-polycythemic myelofibrosis, one with myelofibrosis with myeloid metaplasia, and one with Ph+ chronic myeloid leukemia in blastic phase. Except for the last patient, all patients had been treated with 32P and/or an alkylating agent prior to cytogenetic examination. This is the first report of the 11q21 abnormality in essential thrombocythemia seen at diagnosis.     

Cellular-telephone use and brain tumors

BACKGROUND: Concern has arisen that the use of hand-held cellular telephones might cause brain tumors. If such a risk does exist, the matter would be of considerable public health importance, given the rapid increase worldwide in the use of these devices. METHODS: We examined the use of cellular telephones in a case-control study of intracranial tumors of the nervous system conducted between 1994 and 1998. We enrolled 782 patients through hospitals in Phoenix, Arizona; Boston; and Pittsburgh; 489 had histologically confirmed glioma, 197 had meningioma, and 96 had acoustic neuroma. The 799 controls were patients admitted to the same hospitals as the patients with brain tumors for a variety of nonmalignant conditions. RESULTS: As compared with never, or very rarely, having used a cellular telephone, the relative risks associated with a cumulative use of a cellular telephone for more than 100 hours were 0.9 for glioma (95 percent confidence interval, 0.5 to 1.6), 0.7 for meningioma (95 percent confidence interval, 0.3 to 1.7), 1.4 for acoustic neuroma (95 percent confidence interval, 0.6 to 3.5), and 1.0 for all types of tumors combined (95 percent confidence interval, 0.6 to 1.5). There was no evidence that the risks were higher among persons who used cellular telephones for 60 or more minutes per day or regularly for five or more years. Tumors did not occur disproportionately often on the side of head on which the telephone was typically used. CONCLUSIONS: These data do not support the hypothesis that the recent use of hand-held cellular telephones causes brain tumors, but they are not sufficient to evaluate the risks among long-term, heavy users and for potentially long induction periods.     

A simple method for the quantitation of specific cytotoxic precursors to synrgeneic tumors

Cytotoxic T lymphocytes (CTLs) to EL4, a syngeneic lymphocytic leukemia of C57BL/6 (B6) (H-2^b) mice, were obtained by culturing normal B6 spleen cells with irradiated EL4 tumor cells for 4 days in conical bottom mitrotiter trays. A much lower, though significant, amount of cytotoxicity towards EL4 was observed in B6 spleen cell cultures not stimulated with EL4. The cytotoxic effector cells were shown to be Thy-1⁺ and their cytolytic activity to EL4 tumor cells was inhibited by an anti-H-2^b serum. Cold target competition studies suggest that the B6 anti-EL4 CTLs could discriminate between EL4 tumor cells and a second H-2^b lymphoma, C1498. The converse experiment yielded similar results. Culture conditions limiting for CTL precursors (CLPs) to EL4 were attained by including interleukin 2 (IL2), a lymphokine obtained by stimulating murine or rat spleen cells with concanavalin A, in the culture medium. In the presence of IL2, the CLP frequencies in B6 spleen cells to EL4 and C1498 in antigen-stimulated cultures were 113 and 231 per 10⁶ responder cells, respectively. In non-antigen stimulated B6 spleen cell cultures, the apparent CLP frequencies to EL4 and C1498 were 6 and 33 per 10⁶ responder cells, respectively. In agreement with the cold target competition studies using CTLs generated in the absence of IL2, we found that the CTL clones produced in cultures stimulated with EL4 and C1498 in the presence of IL2 were specific for the stimulating antigen. The specificity of the CTL clones obtained from cultures stimulated with IL2 in the absence of antigen appears to be different from those derived from antigen-stimulated cultures. The potential applications and limitations of this culture system are discussed.     

A modified method for the isolation and determination of brain histamine using Bio-Rex 70

Bio-Rex 70, a weak cation exchange resin, has been used to specifically isolate histamine from rat brain tissue. This method compares favourably to other extraction procedures with respect to selectivity, reproducibility and time taken to perform the procedure. However, because it combines the optimum of these properties, it appears more adaptable for routine laboratory use. Following isolation, histamine is quantified fluorometrically after condensation witho-phthaldialdehyde (OPT). The sensitivity of the procedure allows for the chromatographic isolation, using Bio-Rex 70, of 12.5 ng histamine to give fluorescence twice that of blank. In addition, the use of Bio-Rex 70 enables the selective separation of histamine from fluorescent contaminants such as spermidine. The stability and the reproducibility of the adsorption and elution characteristics of Bio-Rex 70 enables the determination of 30 brain samples in a working day. This method has been applied to determine whole brain and regional brain levels of histamine in control andl-histidine-treated rats. The whole brain level of histamine, which was 50 ng/g, was increased byl-histidine and the highest concentration of histamine was found in the hypothalamus. Since the reliability of existing histamine extraction procedures is questionable, under certain conditions, it is suggested that the use of Bio-Rex 70 is a valuable addition in evaluating the possible physiological role of brain histamine.     

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.     

A specific one step method for the isolation of pancreatic elastase; its use to characterize aortic elastase

Elastase was purified from an acetone-ether powder of porcine pancreas by a one step affinity chromatography procedure on IgG-Sepharose 4B. The IgG was derived from a rabbit immunized with porcine pancreatic elastase and was itself isolated by affinity chromatography on elastase immobilized on the same matrix. The column was calibrated with a known elastase preparation under standardized conditions. Direct isolation of elastase from pancreatic extracts yielded approximately 90 mg from two pancreas. The enzymatic activity and electrophoretic migration in SDS-polyacrylamide gel of the purified elastase were equal to those of the purest commercially available enzymes. Application of this method to aorta of young pigs suggests the presence of active elastase in the aorta.     

A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel

A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors.     

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.     

A simple method for determining relative reward value of brain stimulation

A method for contrasting the reward value of up to four different loci of brain stimulation is described. Animals could insert their snout into any of four holes in the walls of the apparatus, a response at each hole delivering a different current level or locus of stimulation. The response requirement and procedure led to fast acquisition of self-stimulation and choice behaviors. Response rate at the preferred current level at each electrode was also measured. In the majority of cases, rate data would have led to erroneous conclusions about preference.