A versatile two-photon fluorescent probe for ratiometric imaging E. coliß-galactosidase in live cells and in vivo.

We have described the design, synthesis, spectroscopy and biological applications of NI-ßGal, a versatile fluorescent probe to detect E. coliß-galactosidase in live cells and mice sensitively and directly, which holds great promise for its application in biomedical research such as gene therapy for cancer in the future.

IMMUNO-MODULATORY PROPERTIES OF PREBIOTICS EXTRACTED FROM vernonia amygdalina.

BACKGROUND: Vernonia amygdalina, commonly called bitter-leaf, is widely consumed in many parts of Africa, and Nigeria, in particular. The leaf extract has been reported to have antimicrobial, anti-plasmodial, anti-helminthic, as well as prebiotic properties, but its immuno-modulatory effects have not been well-studied, neither have the prebiotics been identified. This study evaluated the immuno-modulatory properties of the aqueous leaf extract and identified the prebiotic components. METHODS: The immuno-modulatory potential was evaluated by monitoring the effects of oral administration of the extract on immunological, haematological and lipid profiles of Rattus norvegicus, while the prebiotic components were identified by thin layer chromatography (TLC), following liquid-liquid fractionation of the extract. RESULTS: Consumption of the extract caused significant increases in CD4+-, white blood cell-, total lymphocyte- and high density lipid (HDL) counts; decreases in low density lipid (LDL) and triglycerides and no significant effect on haemoglobin (Hb) and packed cell volume (PCV) in the blood of test animals. The water-soluble fraction of the extract contained most of the phyto-constituents of the extract and Thin Layer Chromatographic analysis of the fraction revealed the presence of fructo-oligosaccharide and galacto-oligosaccharide prebiotics. CONCLUSION: The results from this study have shown that the aqueous leaf extract of V. amygdalina has positive immune-modulatory and haematologic effects and contains some important prebiotic compounds.

Midgut protease activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

We assayed the relative activities of midgut proteolytic enzymes in individuals of the fourth (L(4)) and fifth (L(5)) instar of Apollo larvae, inhabiting Pieniny Mts (southern Poland). The comparisons between midgut tissue with glicocalyx (MT) and liquid midgut contents with peritrophic membrane (MC) were made. Optimal media pHs of the assayed proteolytic enzymes in P. apollo midgut samples were similar to those of other lepidopteran species. Endopeptidases, as well as carboxypeptidases, digested effectively in alkaline environment, while aminopeptidases were active in a broad pH range. Trypsin is probably the main endoprotease (correlation with caseinolytic activity in MC of L(5) larvae: r=0.606; p=0.004); however, its activity was low as compared with that in other leaf-eating Lepidoptera. This suggests a minor role of trypsin and chymotrypsin in protein digestion in Apollo larvae, probably due to limited availability of the leaf proteins. Instead, due to very high carboxypeptidase A activity in midgut tissue, the larvae obtain exogenous amino acids either directly or from oligopeptides and glycoproteins. High and significant positive correlations between the enzyme activity and glucosidase as well as galactosidase activities strongly support this opinion.

Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells.

Despite successes in animals, cytokine gene expression selectively in human tumors is difficult to achieve owing to lack of efficient delivery methods. Since interleukin (IL)-2-activated natural killer (A-NK) and phytohemagglutinin and IL-2 activated killer T (T-LAK) cells, as previously demonstrated, localize and accumulate in murine lung tumor metastases following adoptive transfer, we transduced them to test their ability to deliver products of genes selectively to tumors. Assessments of transduction efficiency in vitro demonstrated that adenoviral transduction consistently resulted in high (>60%) transduction rates and substantial expression of transgenes such as GFP, Red2, luciferase, beta-galactosidase and mIL-12 for at least 4 days. In vivo experiments illustrated that Ad-GFP transduced A-NK and Ad-Red2 (RFP) transduced T-LAK or mIL-12 transduced A-NK cells localized 10-50-fold more or survived significantly better than mock transduced cells, respectively, within lung metastases than in the surrounding normal lung tissue. Most importantly, mIL-12 transduced A-NK cells provided a significantly greater antitumor response than non-transduced A-NK cells. Thus, adoptive transfer of A-NK and T-LAK cells represents an efficient method for targeting products of genes to tumor sites.

Prevalencia de la enfermedad de Anderson-Fabry en varones con hipertrofia ventricular izquierda / Prevalence of Anderson-Fabry diseases in men with ventricular hypertrophy. AEBM reference 2003 award

La enfermedad de Anderson-Fabry es causa de hipertrofia ventricular izquierda en adultos. En este estudio se pretende analizar la incidencia de la enfermedad deAnderson-Fabry en varones con hipertrofia ventricular izquierda. Se trata de un estudio monocéntrico y prospectivo de 200 pacientes varones con hipertrofia ventricular izquierda. Se realiza la determinación de la actividad de alfa galactosidasa plasmática, confirmándose con la actividad enzimática en leucocitos. Se realizó el diagnóstico de enfermedad de Anderson-Fabry mediante análisis de actividad enzimática plasmática en tres pacientes, en dos de ellos se confirmó el diagnóstico mediante análisis de la actividad enzimática en leucocitos, descartándose dicho diagnóstico en el tercer paciente mediante esta técnica. La hipertrofia ventricular izquierda puede ser una manifestación de la enfermedad de Anderson-Fabry. Se debe tener en cuenta su diagnóstico en todos los pacientes varones con hipertrofia ventricular izquierda, especialmente si es moderada o severa, independientemente de la existencia de posibles causas de ésta (AU)

Potentiation of human estrogen receptor alpha-mediated gene expression by steroid receptor coactivator-1 (SRC-1) in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to reconstruct a human estrogen receptor alpha (ERalpha)-mediated transcription activation system. The level of reporter gene activation was dependent on both the position of the estrogen response element (ERE) relative to the translation start site and the number of EREs in the hybrid promoter. A G400V amino acid alteration in the ERalpha polypeptide decreased sensitivity to 17beta-estradiol (E(2)), demonstrating the hormone responsiveness of ERalpha to be qualitatively and quantitatively similar in yeast and mammalian cells. Coexpression of SRC-1a, a potent stimulator of ERalpha function in mammalian cells, potentiated ERalpha-mediated gene expression over fivefold in a E(2)-dependent manner. Deletion of 56 amino acids at the C-terminal end of SRC-1a resulted in a protein with enhanced ability to potentiate ERalpha-mediated gene expression, which mimics the activity of the same truncation in human SRC-1a as well as the SRC-1e isoform that has the 56 C-terminal residues replaced with a different 14 amino acid peptide. The selective estrogen receptor modulator tamoxifen acted as a weak agonist of ERalpha-mediated gene expression and this weak activity was potentiated by SRC-1. Tamoxifen had no effect on E(2)-induced gene activation in either the presence or absence of SRC-1. In contrast to previously reported yeast-based ERalpha-transactivation systems, the system reported here in which SRC-1 functions as a bona fide coactivator should permit a more thorough dissection of the factors involved in ERalpha-mediated transcriptional activation.

Enzymatic differentiation of Candida parapsilosis from other Candida spp. in a membrane filtration test.

A previously reported enzyme assay on a membrane filter using 4-methylumbelliferyl (4-MU)-N-acetyl-beta-D-galactosaminide, -phosphate and -pyrophosphate as substrates for the differentiation of four Candida spp. has been extended to Candida parapsilosis. The substrate 4-MU-beta-D-glucoside was hydrolyzed by 28 test strains of this species but to a variable extent by seven other yeasts also. For a full enzymatic differentiation of C. parapsilosis from other medical yeasts, a battery of six reactions was required. Of 71 C. parapsilosis positive clinical samples, 4.2% gave a false negative result due to overgrowth by Candida albicans. The present assay is more rapid than a described spectrofluorometric determination of beta-D-glucosidase in a broth, i.e., 9-11 h versus up to >48 h.

Early warning of faecal contamination of water: a dual mode, automated system for high- (< 1 hour) and low-levels (6-11 hours).

There is a recognised need for methods that permit rapid estimation of the sanitary quality of water e.g. during raw water monitoring and emergencies involving water treatment failure or main breaks in a distribution network. In this study, two models for predicting the level of faecal contamination of water were studied. The first format, based on measurement of beta-galactosidase activity by the automated Colifast analyser, detected faecal contamination of high levels, corresponding to > 15 thermotolerant coliforms (FC)/5 mL, in 1-3 h, in a format that allowed for semi-quantification of results. By setting up a cut-off level, the system could be used as an operational tool identifying random increases in faecal contamination during routine raw water monitoring. A second Presence-Absence format was dependent upon the growth of low levels of FC with subsequent detection in the Colifast analyser. 95% of water samples containing 1-15 FC/sample volume showed positive detection after 11 h.

Detection and differentiation between mycotoxigenic and non-mycotoxigenic strains of two Fusarium spp. using volatile production profiles and hydrolytic enzymes.

Volatile profiles and hydrolytic enzyme production by one non-mycotoxigenic and three mycotoxigenic strains of Fusarium moniliforme and F. proliferatum, grown in vitro for up to 96 h on a grain medium at 25 degrees C/0.95 water activity, were examined for differentiation of isolates. After spore lawn inoculation, measurements were made after 48, 72 and 96 h by sampling the head space above cultures with an electronic nose system using a 14 sensor surface polymer array, and by extraction and quantification of hydrolytic enzymes. There was good reproducibility of volatile patterns between replicates of the same treatment. Principal component analysis indicated that discrimination could be achieved between the uninoculated controls, the non-mycotoxigenic strain and the mycotoxin-producing strains for both species after 48 h. The total and specific activity of three out of seven enzymes (beta-D-glucosidase, alpha-D-galactosidase and N-acetyl-beta-D-glucosaminidase) were found to increase significantly in the non-mycotoxigenic when compared with the toxigenic strains of both species after 72 h. Activities of the others (beta-D-fucosidase, alpha-D-mannosidase, beta-D-xylosidase and N-acetyl-alpha-D-glucosaminidase) were not significantly different between strains. The study has shown for the first time that it is possible to differentiate between mycotoxigenic and non-mycotoxigenic strains of such spoilage fungi based on their volatile production patterns using an electronic nose system. These results have significance in the development of methods for the early detection of toxin-producing spoilage moulds in the food industry.

Intercellular Ca(2+) waves induce temporally and spatially distinct intracellular Ca(2+) oscillations in glia.

Mechanically induced intercellular Ca(2+) waves propagated for approximately 300 microm in primary glial cultures. Following the wave propagation, 34% of the cells displayed Ca(2+) oscillations in a zone 60-120 microm from the stimulated cell. The initiation, frequency, and duration of these Ca(2+) oscillations were dependent on the cells' distance from the wave origin but were not dependent on the cell type nor on the magnitude of the Ca(2+) wave. When an individual cell propagated two sequential intercellular Ca(2+) waves originating from different sites, the characteristics of the Ca(2+) oscillations initiated by each wave were determined by the distance of the cell from the origin of each wave. Each Ca(2+) oscillation commonly occurred as an intracellular Ca(2+) wave that was initiated from a specific site within the cell. The position of the initiation site and the direction of the intracellular Ca(2+) wave were independent of the orientation of the initial intercellular Ca(2+) wave. Because initiation and frequency of Ca(2+) oscillations are dependent on the intracellular inositol trisphosphate concentration ([IP(3)](i)), we propose that the zone of cells displaying Ca(2+) oscillations is determined by an intercellular gradient of [IP(3)](i), established by the diffusion of IP(3) through gap junctions during the propagation of the intercellular Ca(2+) wave. Exposure to acetylcholine, a muscarinic agonist that initiates IP(3) production, shifted the zone of oscillating cells about 45 microm farther away from the origin of the mechanically induced wave. These findings indicate that a glial syncytium can resolve information provided by a local Ca(2+) wave into a distinct spatial and temporal pattern of Ca(2+) oscillations.

Numerical techniques and mathematical modelling for CI857-controlled gene expression and cell growth in recombinant E. coli.

Recombinant gene expression, monitored by beta-galactosidase activity, is studied in a pL, pR-CI857 plasmid expression system in temperature-induced E. coli batch cultures. The experimental procedure has been mathematically modelled, and the corresponding parameters are estimated from specific statistical and numerical methods, basically by using a global least-squares procedure under some constraints induced by the model. The numerical techniques proposed in this work act by accumulation of data coming from several runs of the modelled experiment, so that more accuracy is obtained in the parameter estimation. In particular, for the production process, an extra-model parameter depending on an indicator vector is introduced for each run of the experiment in order to globalize the data. The analysis of the data obtained leads to an integrated model for both cell growth and gene expression, which describes an asymmetric dynamics between culture growth and recombinant protein yield, and can serve to predict the maximal value of accumulated gene expression and the time required for it to be achieved at any age of the preinducing cell growth.

Antioxidant vitamins C and E affect the superoxide-mediated induction of the soxRS regulon of Escherichia coli.

The mechanism of activation of Escherichia coli redox sensory protein SoxR is still unclear: a [2Fe-2S] cluster contained in a SoxR dimer is potentially redox-sensitive, but the nature of the signal is unknown. Antioxidant vitamins C (ascorbate) and E (alpha-tocopherol) were used to explore the mechanism of activation of the SoxR protein in vivo. Treating E. coli cells with ascorbate or alpha-tocopherol increased their tolerance to paraquat (PQ, a redox-cycling compound), even in the absence of the soxRS locus, suggesting a radical-quenching activity. When using a soxS'::lacZ fusion, whose expression is governed by activated SoxR, ascorbate and alpha-tocopherol also prevented the expression of beta-galactosidase after PQ treatment. A secondary activity was observed in cells carrying soxR101, a mutation resulting in the constitutive expression of the sox regulon, where the overexpression of soxS'::lacZ was also reduced by ascorbate or alpha-tocopherol treatment. Additionally, different mechanisms of action were revealed as alpha-tocopherol was capable of preventing both PQ and meanadione (MD) lethality, whilst ascorbate prevented PQ lethality but increased MD-mediated cell death. It is proposed that alpha-tocopherol, positioned in membranes, can prevent superoxide-dependent membrane damage; however, water-soluble ascorbate is unable to do so and can even increase the concentration of oxygen radicals reacting with released membrane-associated Fe(II).

Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae.

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.

Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE.

Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed. The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

GM1-gangliosidosis type 1 involving the cutaneous vascular endothelial cells in a black infant with multiple ectopic Mongolian spots.

GM1-gangliosidosis (GM1) is one of the metabolic storage diseases, of which a differential diagnosis requires an array of biochemical assays to determine the enzyme deficiency. This approach is not only time-consuming and costly but also unavailable to most hospital laboratories. However, a presumptive diagnosis of GM1 may be made on the basis of coarse facial feature, foamy endothelial cells in the cutaneous blood vessels and ectopic Mongolian spots, if present. A more definitive diagnosis of GM1 is then made on the demonstration of deficiency of GM1 beta-galactosidase in leukocytes, plasma or cultured skin fibroblasts. Thus, a battery of enzyme tests may be averted.

Spatially localized neuronal cell lineages in the developing mammalian forebrain.

The role of cell lineage in the organization of the cerebral cortex and striatum of the developing rat forebrain was analysed using retroviral-mediated gene transfer to mark the progeny of individual progenitors. Injections around the onset of neurogenesis (embryonic day 14) produced neuronal- and glial-specific clones in the striatum and cortex. The majority of the neuronal clones were restricted to either the deep or superficial layers of the cortex and to either the striatal patch or matrix compartments of the striatum. Moreover, modeling the distributions of the neuronal clones in various ways revealed that grouping the clones into deep vs superficial cortical compartments and patch vs matrix striatal compartments best accounted for the clone distributions. These results suggest that at the onset of neurogenesis there is a heterogeneity of neuronal progenitors within the proliferative ventricular zone.

Epidemiological estimation of Pseudomonas aeruginosa strains isolated from different environments.

117 P. aeruginosa strains have been isolated from hospital material (vascular ward), from ambulatory patients, from lake water samples and from animal environment. The serological characters of the above strains, their phage--typing patterns, their capability of producing ONPG hydrolase were compared in order to find out the strain with identical features responsible for nosocomial infections and also to find endemic infections. There were eleven polyagglutinable strains (isolated from sinks in the vascular ward) which agglutinated with two sera and one strain isolated from lake water. Apart from one exception there were no confirmations of the occurrence of the strains of identical features in different environments. Possible variability within the scope of somatic antigen and the phage typing of microorganism confirm the necessity to use several techniques to carry out studies of epidemiological significance.

[Rapid differentiation method for Shigella and Escherichia coli--application of Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests].

For the differentiation of Shigella from Escherichia coli, Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests were used. A total of 377 Shigella and 124 E. coli strains was examined for each sero- and biosero-type by using these tests. The results were as follows. 1) There were no Shigella strains showing positive reactions for both Indole and ONPG tests. 2) No E. coli strains with Shigella-like characteristics (negative for lysine-decarboxylase, motility and lactose-fermentation tests) showed negative results for both Indole and PGUA tests. 3) The abovementioned strains were classified into twelve types according to the results of these tests. Shigella strains, thus, were differentiated from antigenically Shigella-like E. coli strains. Additional use of these tests together with the conventional methods may valuable for the identification of Shigella strains.