Evaluation of beta-glucuronidase assay for the detection of Escherichia coli from environmental waters.

The new United States Drinking Water Regulations state that water systems must analyze for Escherichia coli or fecal coliforms on any routine or repeat sample that is positive for total coliforms. The proposed methods for the detection of E. coli are based on beta-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide (MUG). This study was conducted to determine whether beta-glucuronidase negative E. coli were present in significant numbers in environmental waters. Two hundred and forty E. coli cultures were isolated from 12 water samples collected from different environmental sources. beta-glucuronidase activity was determined using lauryl tryptose broth with MUG, EC broth with MUG, and the Autoanalysis Colilert (AC) procedure. The isolates were also evaluated by the standard EC broth gas fermentation method for fecal coliforms. The results confirm that assaying for the enzyme beta-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters.     

rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells.

The rha1 gene from Arabidopsis encodes a small GTP binding protein belonging to the Ypt/Rab family. Transgenic Arabidopsis plants containing the promoter region of the rha1 gene fused to the beta-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the root tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl beta-D-glucuronide and a newly developed GUS substrate: Sudan II-beta-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by whole-mount mRNA in situ hybridization. A hypothesis for the function of the Rha1 protein is discussed.     

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.     

Klotho as a regulator of oxidative stress and senescence

The klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. The klotho gene encodes a single-pass transmembrane protein that binds to multiple fibroblast growth factor (FGF) receptors and functions as a co-receptor for FGF23, a bone-derived hormone that suppresses phosphate reabsorption and vitamin D biosynthesis in the kidney. In addition, the extracellular domain of Klotho protein is shed and secreted, potentially functioning as a humoral factor. The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology.     

Klotho--a common link in physiological and rheumatoid arthritis-related aging of human CD4+ lymphocytes

Human CD4(+) T lymphocytes undergo aging-related changes leading to decreased immunity to infections and neoplasms, and to increased frequency of autoimmune diseases including rheumatoid arthritis (RA). Certain changes, observed in the CD4(+) cells of RA patients, resemble those observed during physiological aging, but occur at earlier age. Underlying cellular mechanism(s) of these similarities are so far largely unknown. Here we show that KLOTHO, a beta-glucuronidase gene whose activity changes are associated with aging phenotype, is down-regulated at the mRNA, protein, and enzymatic (beta-glucuronidase) activity levels both in the healthy elderly and especially in RA CD4(+) lymphocytes. Although the exact role of Klotho activity for CD4(+) cell function is unknown, we propose here that it might be involved in anti-inflammatory processes occurring in the young and healthy individuals, but reduced in both healthy elderly and RA patients. To support this hypothesis, we show here that the reduction of Klotho expression and activity in both elderly and patients' lymphocytes occurs in concert with the down-regulation of T cell costimulatory molecule CD28, the latter known to be dependent on increased levels of TNF-alpha. Thus, a common mechanism of KLOTHO down-regulation, but executed at various times in life, may underlie both physiological and disease-related T cell aging. Klotho activity might become a target of anti-RA drug development as well as a tool to help increase the immune system efficiency in the elderly.     

Acceptor-specific glucuronyl transfer catalyzed by beta-glucuronidase.

Highly purified rat liver microsomal or lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) catalyzes the specific transfer of glucuronly residues from phenyl-beta-D-[U-14C]glucuronide to acceptor sugars. Specificity requirements of acceptor sugars are found to be: pyranose structure, 4C1-conformation and equatorial position of C2 and C3 hydroxyl groups or pyranose structure, 1C4-conformation and equatorial position of C3 and C4 hydroxyl groups. The acceptor capacities of 30 monosaccharides and glycosides including di- and tri- saccharides conform to this prinicple. The specificity of the beta-glucuronidase catalyzed glucuronyl transfer is proved by the exclusive formation of beta-glucuronly (1--3)glycosidic linkages. Glucuronly transfer rates increase with increasing donor substrate and increasing acceptor sugar concentration. In the presence of 1 M acceptor sugar the ratio of the transfer rate to the rate of enzymatic hydrolysis is about 2:1. An 'acceptor substrate binding site' on the surface of the beta-glucuronidase molecule which brings the C3 hydroxyl function of the acceptor sugar close enough to the C1 atom of the glucuronyl residue, is postulated.     

Regulation of the Na+/K+ ATPase by Klotho

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.     

Purification and characterization of lysosomal β-glucuronidase from human placenta

1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. beta-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl beta-d-glucuronide and phenolphthalein beta-d-glucuronide as substrates. 4. Only one isoenzyme of beta-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated beta-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental beta-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma beta-glucuronidase was also present in plasma.     

Klotho protein deficiency leads to overactivation of mu-calpain

The klotho mouse is an animal model that prematurely shows phenotypes resembling human aging. Here we report that in homozygotes for the klotho mutation (kl(-/-)), alpha(II)-spectrin is highly cleaved, even before the occurrence of aging symptoms such as calcification and arteriosclerosis. Because alpha(II)-spectrin is susceptible to proteolysis by calpain, we examined the activation of calpain in kl(-/-) mice. m-Calpain was not activated, but mu-calpain was activated at an abnormally high level, and an endogenous inhibitor of calpain, calpastatin, was significantly decreased. Proteolysis of alpha(II)-spectrin increased with decreasing level of Klotho protein. Similar phenomena were observed in normal aged mice. Our results indicate that the abnormal activation of calpain due to the decrease of Klotho protein leads to degradation of cytoskeletal elements such as alpha(II)-spectrin. Such deterioration may trigger renal abnormalities in kl(-/-) mice and aged mice, but Klotho protein may suppress these processes.     

Klotho蛋白在糖尿病肾病中的研究进展

Klotho蛋白是近期发现的和衰老密切相关的蛋白,主要表达于肾小管上皮细胞和脑脉络膜。Klotho蛋白的高表达可以增加机体对氧化应激的抵抗。许多研究证实,在糖尿病肾病中,肾脏klotho的表达降低,并且通过调节磷酸盐代谢、抗氧化应激、抗肾脏纤维化、抗肾小球肥大、抗凋亡、抗炎症等途径保护肾功能。本文就klotho蛋白与糖尿病肾病的关系进行综述,探寻其在糖尿病肾病中的分子生物学机制。     

Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols

Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.     

Association between Decreased Klotho Blood Levels and Organic Growth Hormone Deficiency in Children with Growth Impairment

Objective

Klotho is an aging-modulating protein expressed mainly in the kidneys and choroid plexus, which can also be shed, released into the circulation and act as a hormone. Klotho deficient mice are smaller compared to their wild-type counterparts and their somatotropes show marked atrophy and reduced number of secretory granules. Recent data also indicated an association between klotho levels and growth hormone (GH) levels in acromegaly. We aimed to study the association between klotho levels and GH deficiency (GHD) in children with growth impairment.

Design

Prospective study comprising 99 children and adolescents (aged 9.0±3.7 years, 49 male) undergoing GH stimulation tests for short stature (height-SDS = −2.1±0.6). Klotho serum levels were measured using an α-klotho ELISA kit.

Results

Klotho levels were significantly lower (p<0.001) among children with organic GHD (n = 11, 727±273 pg/ml) compared to both GH sufficient participants (n = 59, 1497±754 pg/ml) and those with idiopathic GHD (n = 29, 1645±778 pg/ml). The difference between GHS children and children with idiopathic GHD was not significant. Klotho levels positively correlated with IGF-1- standard deviation scores (SDS) (R = 0.45, p<0.001), but were not associated with gender, pubertal status, age or anthropometric measurements.

Conclusions

We have shown, for the first time, an association between low serum klotho levels and organic GHD. If validated by additional studies, serum klotho may serve as novel biomarker of organic GHD.     

Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone.

Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.     

Kinetic characterization of recombinant human acidic mammalian chitinase

Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.     

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

Comparative activity of arylsulphatases A and B on two synthetic substrates.

Rat liver and human skin fibroblasts arylsulphatase A and B activities on both 4-methylumbelliferyl sulphate and 4-nitrocatechol sulphate were compared. The intracellular distribution of activity differed markedly when 4-methylumbelliferyl sulphate was used from that observed with 4-nitrocatechol sulphate. No discrimination between control and metachromatic leucodystrophy or mucopolysaccharidosis (type VI) could be achieved when 4-methylumbelliferyl sulphate was used as substrate. These results contrast sharply with those obtained with 4-nitrocatechol sulphate and cast doubt on the validity of 4-methylumbelliferyl sulphate as substrate for the determination of arylsulphatase A and B activities.