Beta-glucuronidase Activity Research Articles

Influence of oxidatively modified LDL on monocyte-macrophage differentiation

Transendothelial migration of peripheral blood mononuclear cells (PBMCs) and their subsequent interaction with the subendothelial matrix lead to their differentiation to macrophages (mphis). To study whether preexposure of monocytes in circulation to modified proteins influences their differentiation to mphis, an in vitro model system using isolated PBMC in culture was used. The effect of modified proteins such as oxidatively modified LDL (ox-LDL), acetylated and non-enzymatically glycated-BSA (NEG-BSA) on the differentiation process was studied by monitoring the upregulation of mphi specific functions such as endocytosis, production of matrix metalloproteinases (MMPs), expression of surface antigen, activity of beta-glucuronidase and down regulation of monocyte specific myeloperoxidase activity. Rate of endocytosis, production of MMPs and beta-glucuronidase activity were significantly greater in cells treated with modified proteins irrespective of the nature of modification. Both CuSO4 ox-LDL and HOCl ox-LDL increased the rate of expression of the mphi specific functions. FACS analysis showed that the rate of upregulation of mphi specific CD71 and down regulation of monocyte specific CD14 were high in cells supplemented with modified proteins. Studies using PPARgamma antagonist and agonist suggest its involvement in CuSO4 ox-LDL induced monocyte-macrophage (mo-mphi) differentiation whereas the expression of macrophage specific functions in cells exposed to other modified proteins was independent of PPARgamma. PBMC isolated from hypercholesterolemic rabbits in culture expressed mphi specific functions at a faster rate compared to normal controls indicating that these observations are relevant in vivo. These results indicate that preexposure of monocytes to modified proteins promote their differentiation to mphis and may serve as a feed forward type control for clearing modified proteins.

The effect of drinking milk containing conjugated linoleic acid on fecal microbiological profile, enzymatic activity, and fecal characteristics in humans

BackgroundThe primary objective was to determine whether consumption of conjugated linoleic acids (CLAs) affected the fecal microbiota composition, fecal enzyme activity or fecal composition.MethodsHuman subjects consumed (1 L/day) cows' milk (4% fat) containing (5 mg/g fat) cis-9, trans-11 CLA (CONT), (32 mg/g fat) cis-9, trans-11 CLA (NAT) and (32 mg/g fat) trans-10, cis-12 CLA and cis-9, trans-11 CLA (SYN) for 8 weeks, in addition to their normal diet. Milk feeding periods were separated by 4 week washout periods. Fecal samples were obtained at the beginning (day 0) and the end (day 56) of each milk feeding period. Fecal samples were analysed for microbiological profile, enzyme activity, pH and short chain fatty acid content.ResultsSamples taken at day 0 and day 56 indicated that the numbers of lactobacilli and bifidobacteria significantly decreased after consumption of all experimental milks; total aerobes, total anaerobes, enterobacteria, and enterococci + streptococci did not change. At day 56, the activities of β-glucosidase, nitroreductase, and urease enzymes had decreased compared to samples taken on day 0 for all treatments. β-glucuronidase activity did not change. Fecal pH and ammonia content did not change.ConclusionIt was concluded that observed changes could have been attributed to increased milk intake; no differences could be attributed to consumption of the different CLAs.

Preventive effect of S‐allyl cysteine sulfoxide (alliin) on lysosomal hydrolases and membrane‐bound atpases in isoproterenol‐induced myocardial infarction in wistar rats

In this study, S-allyl cysteine sulfoxide (SACS) was used to evaluate its preventive effect in isoproterenol (ISO)-induced myocardial ischemia in male Wistar rats. Rats were pretreated with SACS (40 and 80 mg kg(-1)) orally for 5 weeks. After the treatment period, ISO (150 mg kg(-1)) was administered subcutaneously to rats at an interval of 24 h for 2 days. The activities of beta-D-N-acetyl-glucosaminidase, beta-galactosidase, beta-glucosidase, and acid phosphatase increased in serum and heart in ISO-induced rats. In addition, these rats showed a significant (p < 0.05) increase in the activities of beta-glucuronidase and cathepsin-D in serum and heart and a significant (p < 0.05) decrease in their activities in lysosomal fraction of the heart. The activity of Na(+)K(+)-ATPase declined, while those of Ca(2+)- and Mg(2+)-ATPases significantly (p < 0.05) elevated in the heart of ISO-induced rats. Pretreatment with SACS (40 and 80 mg kg(-1)) showed a significant (p < 0.05) effect in all the biochemical parameters studied. The effect at a dose of 80 mg kg(-1) body weight was more effective than that at 40 mg kg(-1) body weight and brought back all the biochemical parameters to near normal levels. Hereby, our study shows the membrane-stabilizing as well as antioxidant effects of SACS in ISO-induced rats.

β‐Glucuronidase activity in cerebrospinal fluid pleocytosis due to urinary tract infection

This study examines the beta-glucuronidase activity in the cerebrospinal fluid (CSF) of patients with urinary tract infection (UTI) and sterile CSF pleocytosis and the feasibility of using these measurements for diagnostic purposes. beta-Glucuronidase activity was measured in the CSF from 22 in each group neonates and infants with UTI and sterile CSF pleocytosis, bacterial meningitis, aseptic meningitis of apparently viral etiology and controls without CSF pleocytosis. The median (range) beta-glucuronidase activity in UTI with sterile CSF pleocytosis was 44.1 (33.2-57.1), whereas in the controls without CSF pleocytosis it was 19.1 (7.0-22.7), in aseptic meningitis of apparently viral etiology it was 26.5 (21.0-30.0) and in bacterial meningitis it was 168 (70.0-1152). The difference between the enzyme activity in the CSF of the patients with UTI and those in the other groups of neonates and infants is significant (p < 0.0001), with no overlapping between UTI and the other groups of children studied. Both the sensitivity and specificity of the activity was 100%. Conversely, there was a broad overlapping of the classic CSF laboratory parameters among the groups of subjects studied. beta-Glucuronidase activity in cell-free CSF discerns, with much greater accuracy than the classic CSF laboratory parameters, sterile CSF pleocytosis due to UTI from that of bacterial and viral meningitis and from control subjects without CSF pleocytosis.

Pediatric Development of Glucuronidation: The Ontogeny of Hepatic UGT1A4

This article reports on the development of UDP-glucuronosyltransferase (UGT) enzyme activity in pediatric livers. The substrates 4-methylumbelliferone (4MU) and trifluoperazine (TFP) were used as probes for general glucuronidation and specific UGT1A4 activity, respectively. The activity of hepatic beta-glucuronidase enzymes was also determined so as to investigate the balance between glucuronide clearance and systemic recirculation. UGT activity toward 4MU reached maximum levels by 20 months of age, whereas the activity of beta-glucuronidase was highest in the neonatal liver and decreased to steady-state adult levels by 4 months. The average V(max) and K(m) values for UGT1A4 in pediatric samples were 151.9 +/- 63.5 pmol/min/mg protein and 14.4 +/- 9.6 muM, respectively. Average V(max) was understandably low because of developmental dynamics, but K(m) was similar to values reported elsewhere. When a constant rate of enzyme development is assumed, maximum activity of UGT1A4 occurs at 1.4 years of age. When the intrinsic hepatic clearance of TFP was scaled with an allometric model, hepatic clearance of TFP by UGT1A4 did not reach maximum levels until 18.9 years of age and scaled results underestimated reported in vivo clearances in adult males. No significant differences in UGT activities or hepatic clearance were observed with gender or ethnicity. The developmental dynamics of most drug-metabolizing enzymes are unknown, and this article contains, to our knowledge, the first description of the development of a single UGT isoform in childhood. Ultimately, work such as this is important for predicting drug responses and for developing and evaluating new medications in children.

Directed Evolution of Beta-galactosidase from Escherichia coli into Beta-glucuronidase

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

Effects of the Inoculation of Burkholderia vietnamensis and Related Endophytic Diazotrophic Bacteria on Grain Yield of Rice

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

Characterization of the new β-glucuronidase from Streptococcus equi subsp. zooepidemicus

Recently, a new gene encoding beta-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37 degrees C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52 degrees C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of beta-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of beta-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified beta-glucuronidase from SEZ glcHis was four times as low as beta-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal beta-glucuronidase from E. coli with E. coli beta-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.

Effect of dietary intervention with different pre- and probiotics on intestinal bacterial enzyme activities

To investigate the influence of different pre- and probiotics on faecal beta-glucuronidase and beta-glucosidase activity, as one of the claimed beneficial effects of pre- and probiotics is the hypothesis that these substrates are able to reduce the production of toxic and carcinogenic metabolites by suppressing specific enzyme activities in the colon. Department of Gastrointestinal Research, University Hospital Gasthuisberg, KU Leuven, Belgium. The effect was evaluated in a randomized, crossover study in 53 healthy volunteers who were randomly assigned to one of five treatment groups. At the start and after a 4-week treatment period, the healthy volunteers collected faeces during 72 h. Lactulose and oligofructose-enriched inulin (OF-IN) were chosen as prebiotics, whereas Lactobacillus casei Shirota, Bifidobacterium breve and Saccharomyces boulardii were selected as probiotics. Two synbiotic combinations were evaluated as well. The enzyme activity was assessed spectrophotometricly. Lactulose and OF-IN significantly decreased beta-glucuronidase activity, whereas a tendency to a decreased beta-glucuronidase activity was observed after L. casei Shirota and B. breve intake. To the contrary, B. breve increased beta-glucosidase levels. Supplementation with the synbiotic did not appear to be more beneficial than either compound alone. No influence of S. boulardii was noted. Administration of lactulose, OF-IN, L. casei Shirota or B. breve resulted in a decrease of the beta-glucuronidase activity, which is considered beneficial for the host.

Identification of Lactobacillus sakei genes induced during meat fermentation and their role in survival and growth.

Lactobacillus sakei is a lactic acid bacterium that is ubiquitous in the food environment and is one of the most important constituents of commercial meat starter cultures. In this study, in vivo expression technology (IVET) was applied to investigate gene expression of L. sakei 23K during meat fermentation. The IVET vector used (pEH100) contained promoterless and transcriptionally fused reporter genes mediating beta-glucuronidase activity and erythromycin resistance. A genomic library of L. sakei 23K was established, and the clones were subjected to fermentation in a raw-sausage model. Fifteen in carne-induced fusions were identified. Several genes encoded proteins which are likely to contribute to stress-related functions. One of these genes was involved in acquisition of ammonia from amino acids, and the remaining either were part of functionally unrelated pathways or encoded hypothetical proteins. The construction and use of isogenic mutants in the sausage model suggested that four genes have an impact on the performance of L. sakei during raw-sausage fermentation. Inactivation of the heat shock regulator gene ctsR resulted in increased growth, whereas knockout of the genes asnA2, LSA1065, and LSA1194 resulted in attenuated performance compared to the wild-type strain. The results of our study are the first to provide an insight into the transcriptional response of L. sakei when growing in the meat environment. In addition, this study establishes a molecular basis which allows investigation of bacterial properties that are likely to contribute to the ecological performance of the organism and to influence the final outcome of sausage fermentation.

Adeno-associated Virus Type 5 Reduces Learning Deficits and Restores Glutamate Receptor Subunit Levels in MPS VII Mice CNS

A major challenge in treating lysosomal storage diseases with enzyme therapy is correcting symptoms in the central nervous system (CNS). This study used a murine model of mucopolysaccharidosis type VII (MPS VII) to test whether pathological and functional CNS defects could be corrected by expressing beta-glucuronidase via bilateral intrastriatal injection of adeno-associated virus type 5 (AAV5betagluc) vectors. After injecting AAV5betagluc, different brain regions expressed active beta-glucuronidase, which corrected lysosomal storage defects. Compared to age-matched littermates, adult MPS VII mice were impaired in spatial learning and memory, as measured by the repeated acquisition and performance chamber (RAPC) assay. AAV5betagluc-treated MPS VII mice improved significantly in the RAPC assay, relative to saline-injected littermates. Moreover, our studies reveal that cognitive changes in MPS VII mice correlate with decreased N-methyl-d-aspartate and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor expression. Importantly, AAV5betagluc delivery restored glutamate receptor levels. Together, these data demonstrate that AAV5 vectors deliver a therapeutically effective beta-glucuronidase gene to the CNS and further suggest a possible mechanism underlying spatial learning defects in MPS VII mice.

Improvement of the pBI121 plant expression vector by leader replacement with a sequence combining a poly(CAA) and a CT motif

To improve expression levels of recombinant proteins in plants, a new leader sequence was designed. Several elements known to enhance gene translation and/or transcription were considered, including the CaMV 35S Inr site, a CT-rich motif often shared by highly expressed plant genes and a poly(CAA) region widespread in tobamovirus and plant leaders. The effect of the synthetic leader on gusA expression was evaluated in genetically modified tobacco plants by measuring the beta-glucuronidase activity and the mRNA level. When compared to the gusA leader of pBI121, the new sequence determined a 8.6-fold and a 12.5-fold increase of enzyme concentration taking into account the whole plant population or the above-average expressors, respectively. Since most pCAMBIA vectors harbour a very short 5'-UTR, identical to a fragment of the pBI121 leader, leader replacement with the sequence herein described is strongly suggested.

Repeated bout effect on the cytoskeletal proteins titin, desmin, and dystrophin in rat skeletal muscle

The aim of this study was to evaluate the effect of repeated bouts of exercise on the cytoskeletal proteins titin, desmin, and dystrophin. Rats were made to run downhill for 90 min 1 or 5 times separated by 14 days. Samples were taken from quadriceps femoris muscle 3, 48, 96 h and 50 days after the last exercise session and detected by quantitative PCR, histochemical stainings, and western blot analyses. Histopathological changes in titin, desmin, and dystophin stainings, an increase in beta-glucuronidase activity (a quantitative indicator of muscle damage), a significant decrease in the relative content of dystrophin, and intramyocellular Evans blue staining (signs of changes in sarcolemmal permeability) observed after one exercise session were attenuated after 5 exercise sessions. Titin mRNA level was not increased after the initial exercise session but was increased after the fifth session. Desmin and dystrophin mRNA levels were increased after the first and fifth sessions with desmin showing a smaller increase after the fifth session compared to the first session. Prior exercise induces adaptation that protects the sarcolemma as well as subsarcolemmal, intermediate filament, and sarcomeric proteins against disruption. Changes in mRNA levels of titin, desmin, and dystophin after an acute exercise session obviously reflect the need of these proteins in the repair process following damage. After five sessions increase in mRNA of studied proteins suggest a strong involvement in continuing adaptation to the increased exercise.

Klotho Gets Around

A remarkable range of biological functions and mechanisms of action have been proposed for the protein Klotho. Klotho is a membrane protein with a large extracellular domain. However, its cytoplasmic domain includes just 11 amino acids, which are thought to be unlikely to mediate its regulatory functions. Lack of Klotho causes shortened life span in mice (but so might lots of deleterious mutations), and overexpression of the protein has been reported to extend life span. This was thought to reflect effects on signaling by insulin and insulin-like growth factor 1. Others have proposed that beta-glucuronidase activity of Klotho modifies the transient receptor potential ion channel 5 (TRPV5), supporting localization and activity of the latter at the plasma membrane. Klotho-deficient mice also share characteristics with mice lacking the fibroblast growth factor 23 (FGF23), such as infertility and decreased bone density; indeed, another proposed action of Klotho is the control of mineral metabolism. FGF23 also regulates mineral metabolism, and the latest action proposed for Klotho is to coordinate with the FGF receptor subtype IIIc [FGFR1 (IIIc)] to create a form of the FGFR that mediates binding of FGF23. Klotho turned up in a screen for molecules that bound in vitro to FGF23. FGF23 is itself unusual in that it functions like a hormone; that is, it is produced in bone but is carried in the circulation to regulate kidney function. Transfection of cells that also expressed FGFR1 (IIIc) with Klotho conferred specific high-affinity binding of FGF23 and allowed activation of associated signaling events. In vitro binding assays showed interaction of the extracellular domains of Klotho with a fusion protein of FGF23 with immunoglobulin Fc region. Injection of mice with a monoclonal antibody to Klotho caused changes reminiscent of those in Klotho knockout animals. Circulating FGF23 appears to act selectively at certain tissues, particularly the kidney, even though most tissues have FGFRs. The authors propose that the presence of Klotho may be the key factor that converts FGFR1(IIIc) to a receptor for FGF23, but they don’t propose a grander scheme to account for the range of reported actions of this intriguing protein. I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, T. Fujita, S. Fukumoto, T. Yamashita, Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444 , 770-774 (2006). [PubMed]

Freshwater molluscs from volcanic areas as model organisms to assess adaptation to metal chronic pollution

Cellular biomarkers of exposure and biological effects were measured in digestive gland of snails (Physa acuta) sampled in sites with and without active volcanism in São Miguel Island (Azores). Metal content in digestive cell lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Lysosomal structural changes (lysosomal volume, surface and numerical densities - Vv(LYS,) Sv(LYS) and Nv(LYS-), and surface-to-volume ratio - S/V(LYS)-) were quantified by image analysis, after demonstration of beta-glucuronidase activity, on digestive gland cryotome sections. Additional chemical analyses (atomic absorption spectrophotometry) were done in the digestive gland of snails. The highest metal concentrations were found in snails from the active volcanic site, which agreed with high intralysomal Vv(BSD). Digestive cell lysosomes in snails inhabiting sites with active volcanism resembled a typical stress situation (enlarged and less numerous lysosomes). In conclusion, the biomarkers used in this work can be applied to detect changes in metal bioavailability due to chronic exposure to metals (volcanism), in combination with chemical analyses.

Effect of ginger on bacterial enzymes in 1,2-dimethylhydrazine induced experimental colon carcinogenesis.

Colon cancer is becoming increasingly common in Asian countries and still remains the second leading cause of cancer death in the United States. Ginger, a natural spice having both antioxidant and antimutagenic property, is known to inhibit chemical carcinogenesis. This study was designed to investigate the chemopreventive efficacy of ginger on the activity of bacterial enzymes in rats induced colon cancer by 1,2-dimethylhydrazine. Twenty milligrams per kilogram body weight of 1,2-dimethylhydrazine was administered subcutaneously once a week for the first 15 weeks and then discontinued. Ginger (50 mg/kg body weight/per day, oral) was given at the initiation and also at the postinitiation stages of carcinogenesis to 1,2-dimethylhydrazine-treated rats. The animals were killed at the end of 30 weeks. The macroscopic findings in the colon and the incidence of tumors were recorded in each group, and the activity of beta-glucuronidase and mucinase was estimated in the tissues and fecal contents of rats. After a total experimental period of 32 weeks (including 2 weeks of acclimatization), tumor incidence was 100% in 1,2-dimethylhydrazine-treated rats. The incidence of cancer as well as the number of tumors in the colon was significantly reduced both in the initiation and postinitiation stages of carcinogenesis on ginger administration. The activities of bacterial enzymes beta-glucuronidase (proximal colon, distal colon, intestines, liver and colon contents) and mucinase (colon and fecal contents) were significantly elevated in 1,2-dimethylhydrazine-treated rats as compared with the control rats. The increase in beta-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in the mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Ginger administration to 1,2-dimethylhydrazine-treated rats significantly decreased the incidence and number of tumors as well as the activity of beta-glucuronidase and mucinase. Thus, ginger has a chemopreventive and anticarcinogenic effect against 1,2-dimethylhydrazine-induced colon cancer by virtue of its ability to lower the activities of the microbial enzymes beta-glucuronidase and mucinase.

The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: comparative promoter activity of six TaLtp genes in transgenic rice

Plant non-specific lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an efficient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1-7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the 1-Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no beta-Glucuronidase (GUS) activity was detected in stems. In flowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther filaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.

Tissue-specific expression of tomato Ribonuclease LX during phosphate starvation-induced root growth

Ribonuclease LX (RNaseLX) from tomato (Solanum lycopersicum L.) belongs to the RNase T2/S-RNase superfamily of plant endoribonucleases and this is a report on the characterization of the RNaseLX gene and its encoded protein as a member of the phosphate starvation response in tomato. RNaseLX gene sequences were cloned by a PCR-assisted approach. RNaseLX promoter sequences contained the conserved binding motif of the transcription factor PHR1 known to mediate phosphate starvation-dependent gene expression. The increase of RNaseLX transcript levels in roots during phosphate starvation correlated with high promoter activity in transgenic plants carrying a PromLX::uidA gene construct and pointed to transcriptional control of RNaseLX expression. Histochemical staining for beta-glucuronidase activity and immunodetection of RNaseLX protein revealed striking RNaseLX expression in main and lateral root tips of phosphate-starved transgenic plants, specifically in epidermal cells, as well as in lateral and adventitious root primordia. Induced RNaseLX expression in roots correlated with stimulated growth and elongation of primary and lateral roots during phosphate deprivation. Phosphate-starvation-induced RNaseLX transcript levels in roots were not modulated by auxin or ethylene. These data indicate that the role of intracellular RNaseLX in the phosphate starvation response is connected with specific RNA turnover processes at the root tip.

Protective role of luteolin in 1,2‐dimethylhydrazine induced experimental colon carcinogenesis

The aim of the present study was to unravel the chemopreventive effect of luteolin on bacterial enzymes such as beta-glucuronidase and mucinase in a colon carcinogenesis model induced by 1, 2-dimethyl hydrazine (DMH). Twenty mg/kg body weight of DMH were administered subcutaneously once a week for the first 15 weeks and then discontinued. Luteolin (0.1, 0.2, or 0.3 mg/kg body weight/everyday (p.o.) was administered in a dose dependent manner at the initiation and also at the post-initiation stages of carcinogenesis to DMH treated rats. The animals were sacrificed at the end of 30 weeks. Colon cancer incidence and the activities of bacterial enzymes beta-glucuronidase (in the proximal colon, distal colon, intestines, liver and colon contents) and mucinase (colon and fecal contents) were significantly increased in DMH -treated rats compared to the control rats. On luteolin administration, colon cancer incidence, number of tumors per rat and the activities of beta-glucuronidase and mucinase, were significantly decreased both in the initiation and post-initiation stages of colon carcinogenesis dependent on the three different doses given. The increase in beta-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating toxins, while the increase in the mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus our results demonstrate for the first time that luteolin, a dietary flavonoid, exerts chemopreventive and anticarcinogenic effects against DMH induced colon cancer.

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.