Releasing of plasminogen-activator from the kidney to the blood (author's transl)]

When fibrinolytic activity in blood samples from various vessels was examined by the dilute-blood-clotlysis-time method (DBCLT), it was found to be noticeably high in the renal venous blood, though the activity was not detected by usual blood clotlysis time method. Plasmin was not detected in any blood samples examined, and the contents of fibrinogen and fibrin (or fibrinogen) breakdown products in the renal venous blood were not significantly different from those in the blood from other vessels. However, the high activity of plasminogen-activator was found only in the renal venous blood. Inhibitors on plasmin and plasminogen-activator (urokinase) were detected in almost the same amount in the blood samples from the various vessels. The amount of the inhibitors was sufficient to inhibit the plasminogen activation by urokinase, whose activity was equivalent to the plasminogen-activator activity in the renal venous blood. These results indicate that the high activity by DBCLT in the renal venous blood was derived from the high activity of plasminogen-activator, which was inactivated by inhibitors in undiluted blood. Plasminogen-activator may be released from the kidney to the blood, and immediately inactivated by the inhibitors in renal vein, and then diluted with systemic blood which contains little plasminogen-activator.     

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Pregnancy-associated esterase in sera of baboons

Baboon serum samples were resolved by starch gel electrophoresis and polyacrylamide gradient gel electrophoresis and stained with naphthol substrates for esterase activity. An esterase that hydrolyzed alpha-naphthyl butyrate in preference to alpha-naphthyl acetate was found in very high activities in some individuals but not others. It migrated just cathodal of the albumin band in starch gels. In polyacrylamide gradient gels, it co-migrated with albumin and had an apparent molecular weight of approximately 65,000 daltons. Electrophoretic analysis by gel electrophoresis of random serum samples from male and female baboons indicated that this esterase was present only in the sera of pregnant baboons. Further investigation of serial samples collected from carefully monitored baboons confirmed that the amount of activity of this esterase was correlated with stage of pregnancy. Therefore, it was named pregnancy esterase (PE). PE was detectable by gel electrophoresis and chromogenic staining techniques as early as day 30 of pregnancy; its activity gradually increased with progressive pregnancy and reached maximum activity near full term (182 days). Soon after parturition, the activity of PE decreased rapidly and was not detected in maternal sera by day 14 postpartum. No evidence of PE was detected in sera of pregnant humans.     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。     

Aromatic L-amino acid decarboxylase activities in human lung tissues: comparison between normal lung and lung carcinomas

We measured the activity of aromatic L-amino acid decarboxylase with L-dihydroxyphenylalanine as a substrate (DOPA decarboxylase) in normal lung tissues and lung tumors obtained fresh at surgery. The activity in control human lung tissues was low and variable: 3.50 +/- 0.42 pmole/min/mg protein (n = 56, mean +/- SE, range 0.01-15), indicating the wide individual variations. Most of small cell carcinoma specimens showed very high activity, as compared with both control lung tissues and with other types of non-SCC lung cancers. Similar results were also obtained in the athymic mice heterotransplants of SCC. High activity was also observed using 5-L-hydroxytryptophan as a substrate (5-HTP decarboxylase) in nine SCC samples. Serotonin was not detected in any control lung tissues, but was detected in all the nine SCC samples, but dopamine was detected only in three out of nine SCC samples.     

Heparin-like activity in uterine fluid.

Uterine fluid was collected from a group of normal patients and a group of patients with menorrhagia. Heparin-like activity was detected in 34 out of 38 samples using an anti-Xa heparin assay. The heparin-like activity in uterine fluid was inhibited by adding the heparin antagonist hexadimethrine bromide to the assay. Concentrations of fibrinogen-fibrin degradation products (FDPs) were measured in five samples of uterine fluid. FDPs in the concentration detected had no effect on the anti-Xa assay. Heparin-like activity was higher in the group with menorrhagia, although the differences were not significant. Heparin-like activity increased throughout the menstrual cycle and decreased during menstruation, suggesting a possible cyclical variation in activity. There was no correlation between mast cell numbers in the endometrium and myometrium and heparin-like activity in uterine fluid and no correlation between the numbers and the stage in the menstrual cycle. In a few patients with intrauterine contraceptive devices (IUCDs) heparin-like activity was increased.     

N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt

N-Acetyl-glucosaminidase activity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12–15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.     

Ultracytochemical detection of guanylate cyclase C activity in alimentary tract and associated glands of the rat. Influence of pH, ATP and the ions Mg2+ and Mn2+

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg²⁺ and Mn²⁺, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg²⁺ or Mn²⁺, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg²⁺ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn²⁺ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg²⁺ and ATP atbreak pH 8 and 7.4.     

Tuberculin purified protein derivative up-regulates the telomerase activity of peripheral blood mononuclear cells from patients with pulmonary tuberculosis.

Telomerase activity was detectable in cells of tuberculous pleural effusions at high percentage. To investigate the possible role of telomerase in the immune function, we examined the proliferating state and the expression of telomerase activity in peripheral blood mononuclear cells (PBMC) from 13 patients with pulmonary tuberculosis (TB) and 13 healthy volunteers in response to tuberculin purified protein derivative (PPD) challenge. Exposure of cells to phytohemagglutinin (PHA) significantly promoted PBMC proliferation during a 6 day-period in both TB patient and healthy volunteer groups. PPD treatment also significantly promoted PBMC proliferation during a 6 day-period in TB patient group, but had no significant effect in healthy volunteer group. During the same period, telomerase activity was detected in every PHA- and PPD-treated samples of the TB patient group. However, the telomerase activity was not detected in PPD-treated samples from healthy donors and all the untreated samples. Our results indicate that the telomerase activity in PBMC could be induced by PPD stimulation in TB patients. Telomerase activity may thus play a permissive role in cell division and clonal expansion of the immune cells in response to TB.     

A rapid, colourimetric assay for cytotoxin activity in Campylobacter jejuni

Abstract Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.     

Protease and phospholipase activities of Candida albicans isolated from vaginal secretions with different pH values

Even though vulvovaginal candidiasis and bacterial vaginosis are seldom simultaneously found, we have detected this association at an above average frequency. Thus, we set out to study the activity of proteinases and phospholipases, virulence factors of Candida albicans, to assess their role in the above mentioned association. Of a total of 70 Candida isolates were retrieved from samples of vaginal secretions analyzed at our Diagnostic Service, 65 were identified as C. albicans (a group of n=26 obtained from clinical samples of pH>4.5 and a group of n=39 from clinical samples of pH=or<4.5). The evaluation of phospholipases activity was performed on malt agar and Sabouraud dextrose agar with the addition of egg yolk as substrate. The proteolytic activity was detected on plates of agar base medium with the addition of bovine albumin serum as substrate as sole nitrogen source. Phospholipases activity was essentially the same in both groups of samples (p=0.2003). Proteolytic activity was detected in 61.5% of the isolates from the group with pH=or<4.5 and in 96.2% in the group with pH>4.5; being the former much higher than the latter (p=0.0001). Based on these results we postulate that the simultaneous occurrence of bacterial vaginosis and vulvovaginal candidiasis could be related to the proteolytic activity but unrelated to phospholipases activity.     

The ontogenic development of innate immune parameters of cod (Gadus morhua L.)

The aim of this study was to monitor the ontogenic development of innate immune parameters of cod (Gadus morhua L.) and to determine the presence of maternal IgM. The general protein composition and enzyme activity was also studied. At intervals, samples were collected of fertilized cod eggs and larvae from 3 days after fertilization until 57 days after hatching. Cell lysates were prepared and analysed by Western blotting using antibodies prepared against cod IgM, the complement component C3 and C-reactive protein (CRP) as well as against cod serum proteins and haemoglobin. Antibodies against salmon cathepsins and against several mammalian proteins of immunological significance were also used. Maternal IgM was not detected but C3 and the closely associated apolipoprotein A-I were present from the time of embryo organogenesis. C-reactive protein was not detected and none of the antibodies against mammalian immune parameters cross-reacted with the cod material. Protein and proteomic analysis showed that the major proteins of the egg samples were vitellogenin derived maternal proteins. Other non-vitellogenin maternal proteins, not yet identified, were also detected in the fertilized eggs. Cathepsin was present in all samples, but other enzyme activity was restricted to larval samples from 4 days after hatching when feeding had commenced. Haemoglobin was not detected until 10 days after hatching.     

Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r² = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10² CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (10² to 10⁸ CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Characterization of plasminogen activator in human cervical cells

Plasminogen activator activity was detected in human gynecologic specimens using a synthetic fluorogenic peptide substrate assay and confirmed by an 125I-labeled fibrin plate assay. Epithelial cells in these samples contain enzymatic activity that biochemically resembles both the well-characterized plasminogen activator, urokinase, and the less-specific plasminogen activator, trypsin. Inhibition of the cervical cell activity by diisopropylfluorophosphate and p-nitrophenyl-p'-guanidinobenzoate demonstrates that, like urokinase and trypsin, this plasminogen activator is also a serine protease. Polyacrylamide gel electrophoresis of plasminogen that had been incubated with cervical cells indicated the same mechanism of plasminogen activation as exhibited by urokinase. We attempted to correlate plasminogen activator activity of each sample with cytomorphologic diagnosis. Three of the four dysplastic samples analyzed showed higher plasminogen activator activity than did the normal samples.     

冬虫夏草蛋白图谱及干燥条件对超氧化物歧化酶活性影响

本文采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和双向电泳（2-DE）技术对3个野生冬虫夏草样本和3个培植冬虫夏草样本进行了比较分析。SDS-PAGE结果显示6个样品的蛋白质分子量主要分布在3-66.4kDa之间,Quantity one软件处理凝胶图谱显示蛋白条带在20-23条之间;采用PDQuest软件对双向电泳图谱处理,共检测到670-936个蛋白点,蛋白质主要分布在等电点为pI 4.5-6、分子量为29.0-66.7kDa和14.3-20.1kDa两个区域。尽管在不同样品之间存在一定程度的蛋白质差异,但是并没有发现野生冬虫夏草和培植冬虫夏草之间有显著差异。以超氧化物歧化酶（SOD）活力为评价指标对新鲜冬虫夏草以及冷冻干燥、20℃阴干和60℃烘干冬虫夏草样品进行比较,发现新鲜冬虫夏草SOD活力最高,冷冻干燥对冬虫夏草SOD活力的影响小于20℃阴干干燥,60℃烘干干燥对冬虫夏草SOD活力破坏最大,结果表明干燥条件对冬虫夏草SOD活性极其重要。     

Developmental Changes in the Activity of Xanthine Dehydrogenase and closely Related Enzyme in Chick Tissues

Xanthine-oxidizing activities in the chick tissues were measured by a couple of assay procedures during development of chick embryo. With the usual assay using pterine and NAD⁺, no detectable level of XDH activity was observed in the liver and little in the duodenum before hatching, whereas an appreciable activity was detected in the kidney of chick embryo. When assayed with xanthine and dichlorophenol indophenol, an XDH-like enzyme activity was significantly detected in the embryonic liver, while no further enhancement of the activity was detected in the kidney and duodenum. Electrophoregrams obtained with samples from various developmental stages, followed by activity staining with tetrazolium dye, supported the above results and revealed that the embryonic XDH-like enzyme is not distinguishable from XDH of adult tissue in molecular size. This XDH-like enzyme, pre-existing in the liver before hatching, however, exhibited no cross reaction with antibody against intact XDH. The nature of this material was discussed in comparison with deflavinated XDH.     

Determination of the concentration and specific activity of acetone in biological fluids

The concentration of acetone dissolved in liver perfusion medium was determined by injection of the sample into a gas chromatograph equipped with a Carbopack/Carbowax-packed glass column. Interference from labile acetoacetate which readily decomposes to acetone was eliminated by treating the samples with NaBH4 prior to the analysis. Acetone was detected and quantified as 2-propanol. Separation of labeled 2-propanol in the sample by high-performance liquid chromatography allowed the determination of its specific activity. These methods make possible the convenient and rapid determination of acetone concentration and specific activity in biological samples.     

Denitrification in deep subsurface sediments

Dissimilatory nitrate reduction (denitrification) in subsurface sediments by indigenous microflora was investigated in samples obtained over a range of depths from 0 to 289 m. Denitrifying activity in sediment samples retrieved from similar stratigraphic horizons at four different sites was determined by measuring the accumulation of N₂O using the acetylene blockage technique. Denitrification was detected in unamended samples which received only prereduced deionized water at almost all depths in all sediments sampled. The surface sediments showed the highest denitrification activity. In the deeper sediments, denitrifying activity was much higher in saturated sandy samples and lower or absent in drier clay samples. Addition of nitrate enhanced denitrification activity in all samples from below the water table down to the maximum depth sampled (289 m), while addition of a carbon (succinate) source in general had no stimulatory effect. These results show that denitrifying microorganisms were present in all of the deep subsurface sediments tested in this study. Furthermore, these results suggest that adequate supplies of metabolizable organic carbon were available to support denitrifying activity. However, denitrification may be limited by inadequate supplies of nitrate in the sediments.