Cirugía Mayor Ambulatoria y Atención Primaria / Ambulatory surgery and primary care

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Anatomía de la próstata en ratones motheaten viable (mev) con mutaciones en el gen de la proteína tirosina fosfatasa SHP-1 / Prostate anatomy in motheaten viable (me v) mice with mutations in the protein tyrosine phosphatase SHP-1

Objetivo: Estudiar la anatomía de la próstata y la vesícula seminal en ratones motheaten viable (mev) con mutaciones en el gen PTPN6 que conlleva una severa reducción en la actividad de la proteína tirosina fosfatasa SHP-1. Los ratones mev homocigotos muestran múltiples anomalías que incluyen inmunodeficiencias, aumento en la proliferación de macrófagos, neutrófilos y progenitores de eritrocitos, disminución de la densidad ósea y esterilidad. Material y método: Se analizó la anatomía macro y microscópica de la vesícula seminal y de la próstata, tanto a nivel macro como microscópico, de 5 ratones mev/mev (homocigotos mev) y 8 ratones wt/wt (tipo salvaje) adultos de 7 semanas. Se ha realizado análisis morfométrico computarizado para medir cambios relativos en el volumen epitelial de los diferentes lóbulos prostáticos. Resultados: Todos los ratones estudiados mostraron órganos genitales (pene, testículos, epidídimos, deferentes) y vejiga normales. La vesícula seminal se encontraba ausente en todos los ejemplares mev/mev analizados, siendo normal y muy llamativa en ratones wt/wt. Las diferentes glándulas que componen el complejo prostático (próstata anterior, ventral y dorsolateral) se encontraron atróficas en ratones mev/mev: próstata anterior 0,4 veces, ventral 0,19 veces, dorsal 0,35 veces y lateral 0,28 veces el tamaño de las respectivas regiones en ratones wt/wt. A nivel microscópico los ratones mev/mev mostraron ductos prostáticos mayores y escasos, acinos severamente atróficos con luces vacías y escaso y suelto componente epitelial formando penachos y pliegues, y cambios hiperplásicos en el estroma fibromuscular. Conclusiones: La próstata de ratones mev/mev muestra signos de diferenciación aberrante y el fenotipo resultante puede estar relacionado con la pérdida de función SHP-1. Las anomalías prostáticas en estos ratones influyen, junto con los defectos de la maduración espermática, en su esterilidad. Estos datos sugieren que SHP-1 desempeña un importante papel en la morfogénesis epitelial prostática

Objective: To study prostate and seminal vesicle anatomy in viable motheaten (mev) mice with mutations in the PTPN6 gene leading to a severe reduction in the activity of protein tyrosine phosphatase SHP-1. Homozygous mev mice exhibit multiple anomalies that include immunodeficiencies, increased proliferation of macrophage, neutrophil, and erythrocyte progenitors, decreased bone density and sterility. Materials and methods: We analyzed macro- and microscopic anatomy of the seminal vesicle and prostate macro- and microscopic anatomy of 5 mev/mev and 8 wt/wt adult 7-week-old mice. Computerized morphometric analysis was performed to measure the relative changes appearing in the epithelial volume of the different prostatic lobes. Results: All mice studied revealed normal genital organs (penis, testis, epididymis, vas deferens) and bladder. The seminal vesicle was absent in all mev/mev individuals analyzed, being normal and very noticeable in wt/wt mice. The different glands that compose the prostatic complex (anterior, ventral and dorso-lateral prostate) were atrophied in mev/mev mice: anterior prostate 0.4 times, ventral 0.19 times, dorsal 0.35 times and lateral 0.28 times those of the respective regions in wt/wt mice. Microscopically, mev/mev mice revealed scarce and large prostatic ducts, acini severely atrophic with empty lumen and scarce loose epithelial component forming tufts and infoldings, and hyperplastic changes in fibromuscular stroma. Conclusions: The prostate of mev/mev mice exhibits signs of aberrant differentiation and the resulting phenotype may be related to the loss of function of SHP-1. Prostatic anomalies in these mice affect, together with defects in sperm maturation, their sterility. These data suggest that SHP-1 plays an important role in prostate epithelial morphogenesis

Prostate anatomy in motheaten viable (me(v)) mice with mutations in the protein tyrosine phosphatase SHP-1.

OBJECTIVE: To study prostate and seminal vesicle anatomy in viable motheaten (mev) with mutations in PTPN6 gene leading to a severe reduction in the activity of protein tyrosine phosphatase SHP-1. Homozygous mev mice exhibit multiple anomalies that include immunodeficiencies, increased proliferation of macrophage, neutrophil, and erythrocyte progenitors, decreased bone density and sterility. MATERIAL AND METHOD: We analyzed macro- and microscopic anatomy of the seminal vesicle and prostate macro- and microscopic anatomy of 5 mev/mev and 8 wt/wt adult 7 week old mice. Computerized morphometric analysis was performed to measure the relative changes appearing in the epithelial volume of the different prostatic lobes. RESULTS: All mice studied revealed normal genital organs (penis, testis, epididymis, vas deferens) and bladder. The seminal vesicle was absent in all mev/mev individuals analyzed, being normal and very noticeable in wt/wt mice. The different glands that compose the prostatic complex (anterior, ventral and dorso-lateral prostate) were atrophied in mev/mev mice: anterior prostate 0.4 times, ventral 0.19 times, dorsal 0.35 times and lateral 0.28 times those of the respective regions in wt/wt mice. Microscopically, mev/mev mice revealed scarce and large prostatic ducts, acini severely atrophic with empty lumen and scarce loose epithelial component forming tufts and infoldings, and hyperplastic changes in fibromuscular stroma. CONCLUSIONS: The prostate of mev/mev mice exhibits signs of aberrant differentiation and the resulting phenotype may be related to the loss of function of SHP-1. Prostatic anomalies in these mice affect, together with defects in sperm maduration, for their sterility. These data suggest SHP-1 plays an important role in prostate epithelial morphogenesis.

Knockdown of protein tyrosine phosphatase SHP-1 inhibits G1/S progression in prostate cancer cells through the regulation of components of the cell-cycle machinery.

SHP-1, a haematopoietic cell-specific tyrosine phosphatase, is also expressed in human prostate. In this study, we report that SHP-1 depletion in PC-3 cells induced by small interfering RNAs causes G1 phase cell-cycle arrest accompanied by changes in some components of the cell-cycle machinery. SHP-1 knockdown increases p27(Kip1) (p27) protein stability, its nuclear localization and p27 gene transcription. These effects could be mediated by PI3K-AKT pathway as SHP-1 interacts with PI3K regulating its activity and p110 catalytic subunit phosphorylation. The increase in p27 protein stability could also because of reduced cyclin-dependent kinase (CDK2) activity. SHP-1 knockdown decreases the CDK6 levels, inducing retinoblastoma protein hypophosphorylation, downregulation of cyclin E and thereby a decrease in the CDK2 activity. However, the codepletion of SHP-1 and p27 does not produce re-entry into the cycle, implying that p27 is not required to maintain cell-cycle arrest induced by SHP-1 depletion. The maintenance of the PC-3 cell anti-proliferative response after p27 loss could be because of mislocalization of CDK2 induced by SHP-1 knockdown. This study shows that SHP-1 depletion promotes cell-cycle arrest by modulating the activity of cell-cycle regulators and suggests that SHP-1 may be required for the proper functioning of events governing cell-cycle progression.

Fine-scale genetic structure and gene dispersal in Centaurea corymbosa (Asteraceae) I. Pattern of pollen dispersal.

Pollen dispersal was characterized within a population of the narrowly endemic perennial herb, Centaurea corymbosa, using exclusion-based and likelihood-based paternity analyses carried out on microsatellite data. Data were used to fit a model of pollen dispersal and to estimate the rates of pollen flow and mutation/genotyping error, by developing a new method. Selfing was rare (1.6%). Pollen dispersed isotropically around each flowering plant following a leptokurtic distribution, with 50% of mating pairs separated by less than 11 m, but 22% by more than 40 m. Estimates of pollen flow lacked precision (0-25%), partially because mutations and/or genotyping errors (0.03-1%) could also explain the occurrence of offspring without a compatible candidate father. However, the pollen pool that fertilized these offspring was little differentiated from the adults of the population whereas strongly differentiated from the other populations, suggesting that pollen flow rate among populations was low. Our results suggest that pollen dispersal is too extended to allow differentiation by local adaptation within a population. However, among populations, gene flow might be low enough for such processes to occur.

[Phosphotyrosine phosphatase SHP-1, somatostatin and prostate cancer]. / Fosfotirosina fosfatasa SHP-1, somatostatina y cáncer de próstata.

We review the mechanisms involved in prostatic growth based on androgens and product of neuroendocrine secretion, with special reference to the role of somatostatin (SS) in the inhibition of neoplastic growth. Our contributions in the field confirm the antiproliferative effect of SS on the prostate is mediated by phosphotyrosine phosphatase SHP-1, that is present in human prostate. This enzyme plays a role in the control of prostatic cell proliferation and in the progression of prostate cancer. Besides, we consider its presence may determine the therapeutic potential of SS in the control of prostate cancer.

Fosfotirosina fosfatasa SHP-1, somatostatina y cáncer de próstata / Phosphotyrosine phosphatase SPH-1, somastostatin and prostate cancer

Revisamos los mecanismos de control de crecimiento prostático basados en andrógenos y productos de secreción neuroendocrina, prestando especial atención al papel de la somatostina (SS) como inhibidor del crecimiento neoplásico. Las aportaciones de nuestro grupo confirman que este efecto antiproliferativo de la SS sobre la próstata está mediado por la fosfotirosina fosfatasa SHP-1, presente en la próstata humana. Esta enzima juega un papel en el control de la proliferación celular prostática y en la progresión del cáncer de próstata. Además, pensamos que su presencia podría determinar el potencial terapéutico de la SS en el control del cáncer de próstata (AU)

We review the mechanisms involved in prostatic growth based on androgens and product of neuroendocrine secretion, with special reference to the role of somatostatin (SS) in the inhibition of neoplastic growth. Our contributions in the field confirm the antiproliferative effect of SS on the prostate is mediated by phosphotyrosine phosphatase SHP-1, that is present in human prostate. This enzyme plays a role in the control of prostatic cell proliferation and in the progression of prostate cancer. Besides, we consider its presence may determine the therapeutic potential of SS in the control of prostate cancer (AU)

Improvement of rheological properties of firm acid gels by skim milk heating is conserved after stirring.

The enhancement of the strength of set acid gels by heating milk was related to rheological parameters (water retention capacity, storage modulus) of corresponding stirred gels. To obtain accurate rheological data from stirred gel it was necessary to maintain a constant granulometry of gel particles and to recognize time after stirring as a contributing factor. Two hours after stirring, the gel exhibited a higher storage modulus when milk was heated above 80 degrees C. A measurement of viscosity of just-stirred yoghurt was sufficient to predict correctly the quality of a stirred gel analysed by viscoelastic measurements. Increased resistance to syneresis of just-stirred gels was related to higher viscosity. The quantity of beta-lactoglobulin (beta-Ig) bound to casein micelles explains the improvement of these gel qualities. We have considered that the structure of the initial firm gel (mesostructure level) was conserved in fragments within the stirred gel. Consequently, the explanation given by various authors for the effect of heating milk on the properties of set gels can also be applied to stirred gels. The same mechanism, described in literature for structure formation of set gels from acidified milk is purposed to explain the role of heating milk on the recovery of gel structure after stirring. The beta-Ig association with casein micelles during heating favoured micelle connections during the acidification. It also favoured the association of gel fragments after stirring during the recovery in gel structure.

Effect of linsidomine on the human radial artery.

The response of the human radial artery to a direct NO donor, linsidomine or 3-morpholino-sydnonimine (Sin 1), in the therapeutic management of peri-operative spasm may increase the patency rates of these grafts in the short, medium and long term. Evaluation of the effects of Sin 1 on the human radial artery is of even greater interest as it has not been published previously. Ninety-six human radial artery rings were studied with two protocols. Rings were mounted in an isolated organ bath between two stainless steel metallic rods connected to stress gauges. Protocol 1 studied the vasorelaxant effect of Sin 1 and nitroglycerin (TNT). Protocol 2 studied the reactivity of the radial artery to the vasoconstrictor agents arginine vasopressin (AVP) and angiotensin II (ANG II). The vasorelaxant effect of Sin 1 on the human radial artery was comparable with that of TNT, but with no tolerance effect. After Sin 1 pre-incubation, the vasoconstrictor effect of ANG II was abolished, whereas AVP induced maximum vasoconstriction similar to that of the control (not statistically significant), but with a shift in the EC50 to higher concentrations, EC50=15+/-20 nM. Sin 1 vasorelaxation of rings precontracted by ANG II was maximal, whereas after contraction by AVP, relaxation remained less than 70%; Sin 1 is a potent vasorelaxant on the human radial artery, which does not exhibit cross-tolerance with nitrates. This compound may be used pre- or post-operatively, and would undoubtedly be of benefit in the peri-operative preparation bath.

Direct vascular actions of methyclothiazide in remodeled mesenteric arteries from hypertensive patients.

In vitro experiments were designed to assess the inhibitory effect of the thiazide diuretic methyclothiazide (MCTZ) on contractile responses to norepinephrine (NE) of mesenteric rings from hypertensive patients and normotensive controls. Arteries were taken from portions of mesocolon from 24 patients: 13 hypertensives and 11 normotensives. Changes in the tension of mesenteric ring preparations were measured isometrically. Histologic studies showed that the arteries from hypertensive patients exhibited 1) a greater media thickness-to-lumen diameter ratio, 2) a smaller lumen diameter, and 3) identical media cross-sectional area as those of normotensive controls. At the physiologic level, the hypertensive and normotensive arteries display similar contractile responses to KCl and NE. Furthermore, our results indicate that MCTZ induces concentration-dependent inhibition of the vasoconstrictor responses to NE. This study provides evidence that hypertension is associated with the remodeling of the human mesenteric artery and that MCTZ is as efficient in inhibiting the contractile response induced by NE on hypertensive than on normotensive arteries.

Synthesis of beta-D-glucosyl- and beta-D-fucosyl-glucoses using beta-glycosidase from Thermus thermophilus.

The thermostable beta-glycosidase from Thermus thermophilus, cloned and overexpressed in Escherichia coli was used to catalyze the transfer of beta-D-glucosyl and beta-D-fucosyl from the corresponding p-nitrophenyl-beta-D-glycopyranosides to a hydroxyl group of glucose in the synthesis of beta-D-glucosyl-D-glucopyranoses and beta-D-fucosyl-D-glucopyranoses. The yields in disaccharides produced under conditions of non-initial velocity were very attractive and the formation of the beta(1-3) linked disaccharides was largely favored. The enzyme could constitute a valuable biocatalyst for the synthesis of disaccharides involving such structures as, for example, Bifidus factors.

Mechanisms of methyclothiazide-induced inhibition of contractile responses in rat aorta.

Methyclothiazide (MCTZ), a thiazide diuretic, inhibits the contractile response induced by norepinephrine in aortic rings from 12-week-old spontaneously hypertensive rats (SHR). Although not modified by indomethacin, this inhibition was attenuated by either mechanical removal of the endothelium or N omega-nitro-L-arginine (NOLA) treatment. These results suggest that the MCTZ effects on the norepinephrine-evoked vascular response are mediated by an endothelium-dependent mechanism involving endothelium-dependent relaxing factor (EDRF)/nitric oxide (NO) release. MCTZ was also found to alter the contractile response induced by the addition of Ca(2+) to a depolarizing solution, and this inhibitory effect was partially abolished by NOLA application. Our data led us to propose that MCTZ relaxes aortic rings, resulting in an endothelium-dependent relaxation phenomenon that could even be reinforced under high-K(+) depolarizing conditions.

Multiple regulation of adenylyl cyclase activity by G-protein coupled receptors in human foetal lung fibroblasts.

The pharmacological profile of adenylyl cyclase activity was analysed in WI-38 human foetal lung fibroblasts. Among various agents that act through G-protein coupled receptors, only the beta-adrenergic agonist isoproterenol stimulated and the tetradecapeptide somatostatin (SRIF, sst) inhibited the enzyme activity. The use of the reverse transcription-polymerase chain reaction (RT-PCR) methodology with appropriate cDNAs allowed us to identify the expression of four subtypes of SRIF transmembrane receptors (sst1-4 but not sst5 receptors) in this cell line. By RT-PCR and immunochemistry techniques, we also demonstrated the expression of stimulatory (alpha(s)) and inhibitory (alpha(i1), alpha(i2) and alpha(i3)) G-protein subunits. The known role of the adenylyl cyclase system in cell proliferation and differentiation mechanisms together with the present analysis of the corresponding regulatory network in fibroblasts of human foetal lung add knowledge on the cell line WI-38 that is widely used as a model system in studying cell growth. The importance of this cell class in normal and abnormal lung function and development reinforces the significance of these results.

Identification of functional somatostatin receptors and G-proteins in a new line of human foetal lung fibroblasts.

A new line (FP) of human foetal lung fibroblasts was analysed for the expression of functional, G-protein coupled somatostatin receptors (SSTR). By means of RT-PCR, we identified the expression of SSTR1, SSTR2, SSTR3 and SSTR4, but not SSTR5, subtypes. The same technical approach evidenced the expression of stimulatory (alphas) and inhibitory (alphai1, alphai2 and alphai3) G-protein subunits. The functionality of SSTR was established from the observation of a dose-dependent inhibitory role of SST upon isoproterenol-stimulated adenylyl cyclase activity, an effect that involves G-protein action. Moreover, the functionality of G-proteins was assessed by means of experiments with forskolin and a nonhydrolysable GTP analogue that showed either Gi or Gs activation in the regulation of adenylyl cyclase. Present results represent a first pharmacological characterization of this new line of human foetal lung fibroblasts. The selective presence of some SSTR subtypes and G-protein subunits in addition to the regulatory network of the adenylyl cyclase pathway are features of recognized involvement in cell growth mechanisms. It is of interest for a cell class widely used to study this topic but also important in lung physiology and pathophysiology.

Direct vascular actions of methyclothiazide and indapamide in aorta of spontaneously hypertensive rats.

In vitro experiments were designed to assess the inhibitory effect of the thiazide diuretics methyclothiazide (MCTZ), the hydrochlorothiazide (HCTZ), and the thiazide-related diuretic indapamide (IND) on contractile responses to norepinephrine (NE) and arginine vasopressin (AVP) of aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Changes in the tension of aortic ring preparations were measured isometrically. MCTZ (10(-4) M) induced endothelium-dependent inhibition of the vasoconstrictor responses to NE and AVP only in aortas from SHR, and the maximal vasoconstrictive effect of NE and AVP was decreased by 59 +/- 11% and 32.3 +/- 13%, respectively. Indapamide (10(-4) M) also induced endothelium-dependent inhibition of the contractile response to AVP in aortic rings from SHR, and the maximal vasoconstrictive effect of AVP was decreased by 33 +/- 5%. In contrast, HCTZ did not inhibit the contractile response to either NE or AVP, even at the highest concentration. This study provides evidence that methyclothiazide and indapamide inhibit the contractile response induced by norepinephrine and/or arginine vasopressin on SHR aortic preparations via an endothelium-dependent mechanism.

[Effect of methyclothiazide on the entry of calcium into vascular smooth muscle cells]. / Effets du méthyclothiazide sur l'entrée de calcium dans la cellule musculaire lisse vasculaire.

The possible involvement of calcium and potassium channels in mediating the vascular actions of methyclothiazide (MCTZ), a thiazide diuretic, was investigated in isolated aortic rings from 12 week-old hypertensive rats. MCTZ (10(-4) M) inhibits the contractile response induced by addition of Ca2+ to a depolarizing solution, the maximal contracture is reduced by 87.16 +/- 6.4%. Furthermore this inhibitory effect was unaffected by charybdotoxine a selective blocker of calcium-activated K+ channels (Kca). This suggesting that MCTZ inhibits voltage-gated Ca2+ channels and blunts the Ca2+ entry into vascular smooth muscle cells. This inhibition was partially attenuated by either mechanical removal of the endothelium or N omega-nitro-L-arginine (NOLA) treatment, suggesting that MCTZ effects are also mediated by an endothelium-dependent mechanism involving endothelium-dependent relaxing factor (EDRF)/nitric oxide (NO) release. Taken together, these observations could point to a role of voltage-gated Ca2+ channels and endothelial release of EDRF/NO in the antihypertensive action of MCTZ.

Somatostatina y cáncer de próstata / Somatostatin and prostate cancer

Cada vez es más evidente que la estructura y función prostática no dependen solamente de andrógenos. Actualmente, se admite que en los trastornos de la proliferación prostática están también implicadas hormonas peptídicas, factores de crecimiento, bucles reguladores autocrinos/paracrinos e interacciones entre el epitelio y el estroma. En este sentido, las células neuroendocrinas han despertado un enorme interés, debido en parte a la relación existente en el cáncer de próstata entre diferenciación neuroendocrina y progresión del tumor. Entre los factores sintetizados y liberados por las células NE se encuentra la somatostatina. Este neuropéptido y sus análogos presentan actividad antineoplásica en gran variedad de modelos experimentales tanto in vitro como in vivo.El efecto antiproliferativo de la somatostatina puede ser indirecto, dada la capacidad de la SS de inhibir la secreción de hormonas tróficas y factores de crecimiento, o directo a través de los receptores presentes en las propias células normales y tumorales. Los análogos de somatostatina han demostrado su eficacia en tumores prostáticos experimentales inducidos en animales, sin embargo los resultados obtenidos en los ensayos clínicos son contradictorios. Aunque la somatostatina y sus análogos son capaces de inhibir la liberación de la prolactina y de la hormona del crecimiento, estudios in vivo e in vitro sugieren que la somatostatina pueda actuar directamente sobre el crecimiento del tumor. En este sentido, aunque es evidente que la próstata tiene receptores para somatostatina, los resultados obtenidos sobre su distribución, los subtipos expresados y su relación con diferentes situaciones patológicas son contradictorios e insuficientes. Por lo tanto, es de esperar que un mejor conocimiento de los mecanismos celulares implicados en el efecto aritiproliferativo de la somatostatina ayude a diseñar nuevos análogos específicos que permitan realizar nuevos ensayos clínicos y valorar con exactitud la eficacia de la somatostatina en el tratamiento del cáncer de próstata. (AU)

Modulation of guanosine triphosphatase activity of G proteins by arachidonic acid in rat Leydig cell membranes.

Previous results from our group have indicated that arachidonic acid decrease cAMP production through a modification of heterotrimeric G proteins. In the present study, we have characterized the high affinity GTPase activity present in Leydig cell membranes and its regulation by fatty acids. The high-affinity GTPase activity, measured as [gamma32P] GTP hydrolysis rate, was both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC50 = 26.7+/-1.1 microM. The existence of only two double bonds in linoleic acid is reflected by a decrease in its inhibitory activity (IC50 = 34+/-2.3 microM). Saturated fatty acids showed no effect at this level. The kinetic analysis as interpreted by Lineweaver-Burk plots, indicated that 50 microM arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decreases in the maximal velocity of the reaction. Arachidonic acid modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addition of arachidonic acid to pertussis toxin-treated membranes had no effect on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in Galphas protein. However, ADP-ribosylation with cholera toxin followed by arachidonic acid treatment led to a further 40% inhibition when compared with cholera toxin treatment alone. These results allowed us to postulate that arachidonic acid inhibits the GTPase activity of Gi protein family. To further analyze the mechanism of arachidonic acid inhibition of GTPase activity, the effect of arachidonic acid on the [35S]GTPgammaS binding was studied. No effect of this fatty acid on GTP binding was found. Combining our previous results with those found here, we can conclude that arachidonic acid maintains Gi proteins in their active state, which in turn inhibit adenylate cyclase and results in decrease cAMP levels.

Kinetic study of a thermostable beta-glycosidase of Thermus thermophilus. Effects of temperature and glucose on hydrolysis and transglycosylation reactions.

A beta-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttbetagly) has a broad substrate specificity towards beta-D-glucoside, beta-D-galactoside and beta-D-fucoside derivatives. The thermostability of Ttbetagly was exploited to study its kinetic properties within the range 25-80 degreesC. Whatever the temperature, except around 60 degreesC, the enzyme displayed non-Michaelian kinetic behavior. Ttbetagly was inhibited by high concentrations of substrate below 60 degreesC and was activated by high concentrations of substrate above 60 degreesC. The apparent kinetic parameters (kcat and Km) were calculated at different temperatures. Both kcat and Km increased with an increase in temperature, but up to 75 degreesC the values of kcat increased much more rapidly than the values of Km. The observed kinetics might be due to a combination of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate could act not only as a glycosyl donor but also as a glycosyl acceptor. In addition, when the glucose was added to reaction mixtures, inhibition or activation was observed depending on both substrate concentration and temperature. A reaction model is proposed to explain the kinetic behavior of Ttbetagly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implicating the existence of a transfer subsite.

Cloning and expression of a beta-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme.

A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a beta-galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a beta-glycosidase (ttbeta-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Ttbeta-gly showed strong identity with those of beta-glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of beta-D-galactoside, beta-D-glucoside and beta-D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-fucoside than for p-nitrophenyl-beta-D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: beta1-3 (100%) > beta1-2 (71%) > beta1-4 (40%) > beta1-6 (10%). Ttbeta-gly is a thermostable enzyme displaying an optimum temperature of 88 degrees C and a half life of 10 min at 90 degrees C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.