Degradation of serum albumin by rat liver and kidney lysosomes

Lysosomes were isolated from the livers and from the kidneys of rats treated or not treated with the cysteine proteinase inhibitor leupeptin, and the levels of the intralysosomal serum albumin of the leupeptin-treated rats were compared with those of the saline-treated control rats. Leupeptin caused an intralysosomal accumulation of albumin in vivo because of its potent inhibition of lysosomal protein degradation. In fact, the lysosomes isolated from the livers and kidneys of leupeptin-treated rats almost completely lost their ability to degrade rat albumin in vitro. These findings show that the lysosomes are subcellular sites of the degradation of unlabeled serum albumin in these tissues. They also suggest that cysteine proteinases sensitive to leupeptin are involved in the lysosomal degradation of albumin. Albumin was degraded by total lysosomal enzymes in vitro. It was also degraded by the lysosomal extract being devoid of cathepsins H and J, prepared from rat kidney. The degradation of albumin by total lysosomal enzymes in vitro was greatly suppressed by a cysteine proteinase inhibitor, cystatin alpha, with no inhibition of cathepsins B and L. It was slightly suppressed by N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prol ine (CA-074), a selective inhibitor of cathepsin B, and by pepstatin, an inhibitor of cathepsin D, whereas it was markedly suppressed by a combination of cystatin alpha and either CA-074 or pepstatin. These and associated findings show that cystatin alpha-sensitive cysteine proteinase(s), which is distinct from cathepsins B, H, L, and J, and cathepsins B and D are involved in the lysosomal degradation of albumin.     

Mechanism of the growth-promoting effect of serum albumin on concanavalin A-activated lymphocytes: protective effect of the plasma proteins

The concentration of concanavalin A (Con A) required for optimal growth of lymphocytes is 10 to 20 times greater in the presence of plasma than in the presence of serum albumin (BSA). This shift in Con A requirement is mainly caused by Cohn fractions III and IV, which bind and probably remove free Con A. Aymphocytes, once they are activated by Con A, lose all proliferative activity unless protected by certain proteins. Cohn fraction VI and beta-lactoglobulin were found to be most effective in protecting the cells. The protective proteins do not promote growth; only BSA promotes growth of Con A-activated lymphocytes. The mechanism of the growth-promoting effect of BSA on Con A-activated lymphocytes was investigated. The continous presence of BSA in the culture medium durdly reduces, whereas protein-free medium abolishes, growth. The mechanism of the growth-promoting effect of BSA is not concerned with regulating the uptake of Con Aby lymphocytes. Red blood cells, crenated by washing in protein-free medium, revert immediately to their normal globular shape by the addition ofBSA or FAFBSA, whereas the protective proteins fail to do so, indicating a direct effect of BSA on the external surface of the erythrocyte membrane and presumably also of the lymphocyte. The hypothesis is proposed that BSA promotes growth by conveting total structural integrity to the cell surface membrane of Con A-treated lymphocytes.     

An improved method for detecting passive pills

One of the principal disadvantages of the passive pill as a telemetric method for measuring various physiological parameters has been its resticted range. The reasons for the restricted range with existing detection methods are discussed. An improved method using a locking spectrometer based on third-order phase-sensitive detection is described and its performance is assessed. A significant increase in the usable range of a high sensitivity passive pill is obtained.     

混匀矿粉采样技术的改进与应用

通过对混匀矿粉采样技术的改进，解决了以往混匀矿粉采样缺乏代表性的难题，实现了混匀矿粉按预算成份组织生产的目的，为其他散状物料的采样提供了一定的经验．     

An improved UV-spectrophotometric method for routine barbiturate monitoring

Clinical tests, for instance pupillometry, Valsalvatest, and orthostatic tests show evidence of sympathicopathy in chronic renal failure (GFR: less than 30 ml/min). Hemodialysis does not improve the intensity or frequency of the alterations. Toxic effects of uremic toxins on the autonomic nervous system can be supposed.     

An improved method for chromosome counting in maize

An improved method for counting chromosomes in maize (Zea mays L.) is presented. Application of cold treatment (5C, 24 hr), heat treatment (42 C, 5 min) and a second cold treatment (5C, 24 hr) to root tips before fixation increased the number of condensed and dispersed countable metaphase chromosome figures. Fixed root tips were prepared by the enzymatic maceration-air drying method and preparations were stained with acetic orcein. Under favorable conditions, one preparation with 50-100 countable chromosome figures could be obtained in diploid maize using this method. Conditions affecting the dispersion of the chromosomes are described. This technique is especially useful for determining the somatic chromosome number in triploid and tetraploid maize lines.     

An improved assay method for fibroblast gelatinolytic enzyme

A useful gelatinolytic enzyme assay for fibroblasts, utilizing a novel sample preparation method for collagenase with dithiothreitol (DTT) treatment to inactivate endogenous collagenase inhibitors, was developed using soluble fluorescein isothiocyanate (FITC)-labeled gelatin. The substrate, gelatin was prepared by heating commercially available FITC-labeled type I collagen. The denatured collagen was cleaved with purified trypsin and partially purified fibroblast gelatinase, and the digested FITC-fragments were measured fluorometrically. The intensity of the fluorescence was in proportion to the reaction time and enzyme concentration. Both enzyme activities were measurable within the nanogram range of enzyme preparations. The enzyme activity was detected after 4-aminophenylmercuric acetate (APMA) treatment which was completely inhibited by metalloproteinase inhibitors, but not by serine- and cysteine-proteinases' inhibitors. Conditioned media of human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) were separately treated with DTT prior to the enzyme assay, and then the assay was performed in the presence of APMA. The enzyme activities of PLF and GF were 106- and 55-fold higher than those of the conventional gelatinase assay which was carried out without DTT treatment. This assay method allowed the measurement of gelatinolytic enzyme activity when tissue inhibitors of metalloproteinases were present in the fibroblast culture medium.     

An improved automated fluorimetric method for determination of histamine

The automated continuous flow system for the extraction and fluorimetric analysis of histamine based on the principle of Shore et al. (1959) has been improved. With lower consumption of reagents and further simplification of the working conditions, histamine can be determined quantitatively in a routine fashion in aqueous samples, with or without protein content, up to a concentration of approximately 0.1 ng/ml. The rate of analysis is 30 samples/h.     

An improved filter-disc assay for 3H-DNA and serum deoxyribonucleases

1. The filter-paper disc assay for tritium-labeled nucleic acids was modified to reduce washing and manipulation. Evaluation of the procedure indicated greatly enhanced counting efficiency for tritium, complete removal of nuclease digestion products and retention of even nanogram levels of radioactive DNA on the discs in the presence of protein. The modified assay was quite sensitive and had a coefficient of variation of 5.2%. Quite low concentrations of DNase I and endogenous human serum nuclease activity were readily measured by the improved technique, 22 sera yielding a mean value equivalent to about 0.5 ng DNase I/ml serum. We are now using the disc assay to follow serum DNA and DNase levels in patients with autoimmune disorders and various types of cancers.     

An improved method for coronary sinus blood flow monitoring by intravascular Doppler technique

In order to obtain accurate measurements of coronary sinus blood flow (CSBF), a new catheter (7 French) with a radiopaque, flexible, and basket-shaped tip was developed for guiding a standard 3 Fr Doppler catheter in the coronary sinus (CS) in man. The radiopaque "basket" tip of the catheter allows the operator to stabilize the position of the Doppler transducer in the center of the CS and to accurately measure the CS internal diameter radiologically. CSBF was calculated as the product of CS cross-sectional area by mean CSBF velocity. Doppler-derived CSBF values at rest and during handgrip were compared with those obtained by the local thermodilution technique in 16 patients undergoing diagnostic coronary angiography. During handgrip, mean CSBF increased from 154+/-23 (rest) to 299+/-34 mL/min by the Doppler method and from 148+/-22 to 288+/-32 mL/min by the thermodilution technique. A good correlation (r = 0.86) between the CSBF values with the two techniques was observed. The authors conclude that the intravascular Doppler technique associated with the use of the basket guide catheter provides an accurate and simple tool for monitoring CSBF in patients.     

An improved assessment method for the temperature dependence of yield strength values

In Europe work is in progress to establish new standards for materials and it is very important that accurate data are used as a basis for property values in these standards. In addition reliable evaluation methods must be employed when the values are derived. A systematic evaluation method for yield strengths at elevated temperatures has been developed which is a modification of the ISO 2605/111 (ENV22605-3) procedure. The method has been applied to a number of steel types and has proven to provide values in good accordance with experimental data. Comparisons with existing national and international standards showed the importance of using experimental values when establishing new standards. The method is intended for use in the standard developments by the European Committee for Iron and Steel Standardisation (ECISS).     

黑芥子酶提取及活性测定方法的改进

以拟南芥为材料,对以往黑芥子酶的提取方法和活性测定方法进行了改进,通过实验优选出最佳的提取和活性测定条件.改进后的方法提取样品量只有150 mg,提取缓冲溶液1 mL,离心回收上清作为酶液;3 mL反应体系溶液(含0.2 mmol/L黑芥子硫苷酸钾)置于1 cm光路石英比色杯中,37℃条件下平衡后,向反应体系溶液中添加150μL酶液,预反应3min,而后在227 nm波长下测量吸光度5 min(反应时间),计算酶活.改进后的方法取样量小,操作较为简便,适于较多样品的平行测定.     

A method for the continuous purification of proteins by affinity adsorption

The Food and Drug Administration (FDA) must manage the delicate balance between speed and safety in the evaluation and approval of new drugs. To explore how these competing obligations might be formalized with respect to the AIDS epidemic, we present a simple decision-theoretic optimization model of the clinical trials process against a backdrop of HIV transmission. Our framework sheds light on such issues as the economic consequences of decisions, the potential savings of a new therapy, the costs to society of delay, the value of better information, and how and when to undertake a clinical trial. We believe that this article represents a first effort to unravel the tangled web of epidemiological, economic, and statistical considerations that plague this policy issue.     

Enhancement of phytohemagglutinin and concanavalin A responses of splenic lymphocytes by adherent cell removal

Removal of glass wool-adherent cells resulted in an enhancing effect on DNA synthesis as measured by increased [3H]-thymidine uptake by normal swine spleen cells stimulated with phytohemagglutinin or concanavalin A (Con A). Depletion of nylon-adherent cells caused decreased DNA synthesis in response to Con A, except at high doses of Con A, whereas similar cultures stimulated with phytohemagglutinin showed enhanced DNA synthesis throughout the dose range employed. Titration experiments revealed that enhancing effects of adherent cell removal are most pronounced at higher cell and mitogen concentrations. Glass wool was more efficient than nylon in the enhancement of mitogen-induced DNA synthesis.