Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Synthesis of tritiated rat insulin with high specific activity: A method for radiolabeling the active site of insulin

Insulin was synthesized by rat islets from tritiated amino acids under conditions designed to achieve high specific activity. Islets were isolated by the collagenase method. Stores of unlabeled insulin were depleted by culturing them for 40 hours in the presence of 3-isobutyl-1-methylxanthine. The islets were then incubated for 22 hours in the presence of [³H]Isoleucine or [³H]Phenylalanine. These amino acids were chosen because they are specific markers from the A and B chains of insulin respectively. Labeled insulin was extracted from the islets and purified by gel filtration. Its biological activity was indistinguishable from monoiodinated insulin as assessed by binding to receptors on cultured human lymphocytes and by precipitation by anti-insulin antibodies. The specific activity was (18 Ci/mmole) and (37 Ci/mmole) for [³H]Ile and [³H]Phe insulin respectively.     

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. ³H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.     

人胰腺细胞培养及胰岛素的分泌

人胰腺细胞培养及胰岛素的分泌王石泉,汤国枝,张鹤云,李敏意,金以丰（南京大学生物化学系,南京２１００９３）胰岛β细胞的体外培养获得胰岛素已有报道,但大多采用新生大鼠胰腺,且β细胞成活率低,分泌量少,还处在研究阶段［１－４］．本实验采用人胰腺细胞做较大...     

Preparation of 125I-labeled secretin of high specific radioactivity.

A simple and rapid method of preparing ¹²⁵I-labeled secretin of high specific radioactivity has been developed. Synthetic porcine secretin was iodinated with Na[¹²⁵I] by a modification of the chloramine T method. Purification and separation of labeled from unlabeled secretin was achieved by chromatography on SP-Sephadex C-25 column. The labeled secretin possessed specific radioactivity as high as 500–550 μCi/μg.     

Hemolymph lipids and flight activity in Helicoverpa zea (Boddie) (lepidoptera: Noctuidae)

In this study the flight activity of female and male Helicoverpa zea (Boddie) moths was observed and compared to hemolymph lipid concentrations. The major male and female H. zea flight activity occurred between simulated dusk (1700) and dawn (0300). Male flight activity was up to 7 times greater than females through 6 days after eclosion except for the 1st day (0.8 times). Females had a unimodal pattern of flight activity, peaking between dusk and 2 h later. Males had a bimodal pattern; one between dusk and 2 h later, and another 3 h after dusk, continuing for h. Prior to dusk, total neutral hemolymph lipids (neutral) of H. zea day 4 moths was 64 μg/μl for males and 48 μg/μl for females. Typical lipid composition in day 4 males prior to flight was 1,2-diacylglycerides (DG) (50% w/w), triacylglycerides (TG) (35%), cholesterol esters (2%), and less than 1% monoacylglycerides and cholesterol. The remainder consisted of free fatty acids (<0.5 μg/μl), and various uncharacterized phospholipids and lipophilic compounds. Hemolymph DG concentration patterns were similar between day 4 males and females, were highest in both sexes prior to, during, and after flight (approximately 32 μg/μl), and then decreased steadily throughout the flight period to approximately 16 μg/ml as flight ceased. Hemolymph TG were lower than DG, but followed the same pattern except at 2100 and 2300. In day 4 males between 2100 and 2300, TG increased to 33 μg/μl which was when DG was lowest (15 μg/μl) and their flight activity was highest. Hemolymph DG decreased (26 to 20 μg/μl) in day 4 females between 2100 and 2300 as TG remained fairly constant (18 μg/μl).     

Radioimmunoassay specific for amino (N) and carboxyl (C) terminal portion of parathyroid hormone.

A radioimmunoassay specific for the amino (N) terminal portion of the parathyroid hormone (PTH) molecule (N-PTH radioimmunoassay) has been developed by iodinating synthetic 1-34bovine PTH (1-34bPTH) and using commercially available bPTH antiserum. A radioimmunoassay specific for the carboxyl (C) terminal (C-PTH radioimmunoassay) has been carried out by adding enough amount of 1-34bPTH to the PTH radioimmunoassay system. The data obtained from N- and C-PTH radioimmunoassay were compared with those obtained from the PTH radioimmunoassay. It was observed that plasma levels of N-PTH, indicating biologically active PTH, were only one 8th to 32th to those of PTH and those of C-PTH were almost equal to those of PTH. These data corresponded well with those reported previously by using the antiserum specific for each terminal of the PTH molecule from the other laboratory. The half life of plasma N-PTH and C-PTH determined following the removal of parathyroid adenoma was less than 10 min and about 45 min respectively. These data indicate that the N-PTH radioimmunoassay can be done by iodinating 1-34bPTH and using commercially available antiserum.     

鱼类对藻类消化吸收的研究 (Ⅰ)白鲢对斜生栅藻的消化与吸收

斜生栅藻对32磷有很强的吸收积累能力,吸收率在98%以上。白鲢对用3种不同强度的32磷标记的斜生栅藻中的32磷也有较高的摄取率,平均在43.59—59.78%之间。32磷在白鲢各组织中积累和分布的比放射性(specific radioactivity)大小顺序为:肠肝骨肉脑。白鲢各组织的总放射性(total radioactivity)大小顺序为:肉肠骨肝脑,肌肉中32磷的含量占各组织中32磷的48—70%,说明白鲢能消化吸收斜生栅藻。       

Solid phase lactoperoxidase radioiodination of microgram quantities of protein mixtures for electrophoretic analysis.

A technique for iodinating microgram quantities of heterogeneous protein mixtures, permitting the visualization of individual polypeptide species following electrophoretic separation, has been developed. Specific activities between 1 and 3 × 10⁵ cpm/μg protein are routinely attained with samples as small as 0.5 μg protein. Banding profiles obtained by radioautography are comparable to those determined by Coomassie blue staining; however, 100-fold less material is needed. The presence of carrier molecules such as polyglutamic acid does not interfere with either iodination or electrophoresis in amounts as high as 1 mg.     

The simultaneous determination of the products of estrogen 2- and 4-hydroxylase action; the use of high-performance liquid chromatography with electrochemical detection

Both trace-labeled and high-specific activity ¹²⁵I-labeled derivatives of hexadecapeptide gastrin (G) were prepared by reaction with the iodinated form of the imidoester, methyl p-hydroxybenzimidate. Reaction conditions for preparation of trace-labeled iodinated imidoester gastrin (IIE-G) were: excess imidoester to G (IIE:G, 20:1), pH 9.2, and a reaction time of 24 h. Following purification by gel filtration and silica gel chromatography, an IIE-G component was isolated which appeared homogeneous on thin-layer chromatography and retained the same biological and immunological properties as unmodified G. Somewhat different conditions were necessary to prepare high specific activity iodinated imidoester gastrin (IIE¹-G). These included reducing the volume (20 μl) and pH (7.5) at which the imidoester was iodinated and adjusting the concentrations of reactants to the same molar amounts as 5 mCi of carrier-free ¹²⁵I. Sufficient amounts of IIE¹-G were obtained by reversing the ratio of G and IIE¹ and reacting with a G excess (GIIE¹, 10:1). The purified IIE¹-G had a specific activity exceeding 1500 μCi/nmol and was used to establish a specific and sensitive radioimmunoassay for gastrin.     

不同剂量糖皮质激素治疗老年哮喘急性发作的对比研究

目的:探讨不同剂量吸入型糖皮质激素(ICS)治疗老年哮喘急性发作的疗效及安全性分析。方法:将108例老年哮喘急性发作患者随机分为A组(34例)、B组(38例)和C组(36例),分别吸入布地奈德200μg/d、400μg/d、800μg/d;治疗3个月后再随机分为低剂量组(200μg/d,n=)和高剂量组(400μg/d)进行维持治疗,并随访观察12个月。结果:治疗3个月后,三组的临床症状评分、肺功能较治疗前均有显著改善,差异有统计学意义(P<0.05);B组、C组治疗后的临床症状评分、肺功能及症状消失时间均显著优于A组,而B组、C组之间差异无统计学意义(P>0.05);A组和B组不良反应的发生率分别为5.9%,10.5%,显著低于C组30.6%,差异有统计学意义(P<0.05);维持治疗期间,低剂量组复发率为21.2%,高剂量组为20.4%,差异无统计学意义(P>0.05)。结论:老年哮喘急性发作患者给予ICS 400μg/d治疗剂量及200μg/d维持剂量即可取得较好的治疗效果,可减少不良反应,提高患者的耐受性。     

The hippocampus and septal area as primary target sites for corticosterone

—Two groups of totally adrenalectomized male rats (220-260 g) were injected intraperitoneally with different amounts of [1,2-³H] corticosterone. One group received 1 μg of hormone (50 μCi/μg), and the other received 20 μg (2·5 μCiμg). All animals were decapitated at 15 min and various tissues including the pituitary, cerebellum, pons-medulla, cerebral cortex, hypothalamus, septal area, and hippocampus were assayed for radioactivity and protein content. In the 1-μg group, the hippocampus contained more cpm/mg of protein (P<0·05) than all the other areas including the hypothalamus. The septal area and pituitary gland values were higher (P<0·05) than all the other areas except the hippocampus. In the 20-μg group there was a significant reduction (P<0·05) in the values (cpm/mg of protein) for the hippocampus, septal area and cerebral cortex, but no change in the values for the pituitary, hypothalamus, and pons-medulla. These data suggest that the hippocampus and septal area are primary target sites for corticosterone because they take up more hormone than other nervous tissue loci and demonstrate a limited capacity for hormonal uptake. The pituitary gland tends to concentrate the hormone but does not demonstrate a limited capacity for uptake. The hypothalamus does not concentrate the hormone or demonstrate a limited capacity for uptake, and the data suggest that it is not a primary target site for corticosterone.     

Assay of proteolytic enzymes by the fluorescence polarization technique.

Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB³H₄, followed by separation of the 2 → 3 and 2 → 6 isomers of [³H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The K_m was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases.     

Iodination of mouse egf with chloramine T at 4°C: Characterization of the iodinated peptide and comparison with other labelling methods

Abstract

A modified Chloramine T labelling procedure was used to iodinate mEGF in order to perform radio-receptor assays. The reaction was conducted at 4°C with l µg Chloramine T only. The tracer obtained was characterized by its maximal binding, specific activity and binding properties compared with the native peptide. Fast Liquid Protein Chromatography was performed to analyse the homogeneity of the preparation and membrane extracts from A431 cells were used to purify the tracer. The modified Chloramine T procedure was compared with two other methods: the classical Chloramine T iodination and the labelling procedure using Enzymobeads.

The modified Chloramine T procedure is reproducible, provides labelled mEGF with high binding capacity (65 to 80% with canine placental membrane extracts) and high specific activity (351 ± 107 µCi/µg mEGF) and seems to preserve the binding properties of the native peptide.     

Effect of Chemical Constituents from Syzygium cumini (Myrtaceae)on Glucose Uptake in Insulin-resistant L6 Cells

Eight known compounds isolated from the roots of Syzygium cumini were evaluated for their ability to enhance the glucose consumption in insulin-resistant L6 muscle cells induced by high concentration-insulin and glucose . All of compounds significantly enhanced the glucose consumption of insulin-resistant cells in the presence and absence of insulin. The glucose consumption was increased 17.35% and 51.11% by compound 1 ( friedelin) at a concentration of 10 μg ml^-1 without insulin and by compound 8 (5 , 7 , 3′, 4′, 5′-pentahydroxyflavone) at a concentration of 0 . 1μg ml - 1 with insulin, respectively .     

Quantitative Studies on the Growth of Allogromia laticollaris (Foraminifera)

SYNOPSIS. The growth and reproduction of Allogromia laticollaris was studied. More schizozoites were generally produced in mixtures of food organisms than on single algal foods. In the presence of moderate numbers of bacteria, cultures with Phaeodactylum tricornutum, Chlorococcum sp., Nannochloris sp., and an unidentified chlorophyte (BL-1), added singly, were also highly productive. Schizogony was the dominant asexual form of reproduction. Binary fission and cytotomy also occurred in bacterized otherwise unfed controls. ³⁵S and ³²P are convenient labels for measuring growth of A. laticollaris when introduced into the system in the range of 1 × 10⁴ - 1 × 10⁵ dpm/ml (³²P specific activity ～ 2.03 MCi/g; ³⁵S specific activity ～ 95 μCi/g). Small allogromiids grew faster than did larger ones. By means of the Taylor series modification of the classical least-squares method, a continuous life-cycle representation was calculated for A. laticollaris for the conditions of the experiment. Four points of cell volume growth were maxima for reproduction: 1.0 × 10⁷μ per organism for curve I; 2.2 × 10⁷μ³ and 1.2 × 10⁷μ³ for curve II; and 6.7 × 10⁷μ³ for curve III.     

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>10⁸ cts/min per μg) in vitro using deoxynucleoside [α-³²P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.     

高分辨率熔解曲线法检测CYP4A118590T＞C单核苷酸多态性

目的 建立CYP4A11 8590T＞C单核苷酸多态性（single nucleotide polymorphism,SNP）的高分辨率熔解曲线（high resolution melting,HRM）检测方法.方法先采用温度梯度PCR,确定适宜的退火温度;再利用正交试验,优化引物、DNA模板量和Mg2＋量,最终确定PCR反应体系和反应条件.通过对607例无血缘关系的受试者基因组DNA进行HRM分析,并随机选择50例产物测序.结果 引物最适退火温度为57.8 ℃;PCR最佳反应体系为20 μl,包括2×conc dNTP mix 10 μl,上下游引物（10 μmol/L）各0.5 μl,DNA溶液（30 ng/μl）1.0 μl,Mg2＋（2.5 mmol/L）1.5 μl和灭菌水6.5 μl.607例受试者中CYP4A11 8590TT、TC和CC基因型频率分别为54.7 %、37.6 %和7.7 %.结论该正交试验优化的HRM技术可用于检测CYP4A11 8590T＞C单核苷酸多态性,且其分析结果和测序结果一致.     

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred μg of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.