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1.
Biosensors (Basel) ; 14(5)2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38785720

RESUMEN

Whole-cell biosensors could serve as eco-friendly and cost-effective alternatives for detecting potentially toxic bioavailable heavy metals in aquatic environments. However, they often fail to meet practical requirements due to an insufficient limit of detection (LOD) and high background noise. In this study, we designed a synthetic genetic circuit specifically tailored for detecting ionic mercury, which we applied to environmental samples collected from artisanal gold mining sites in Peru. We developed two distinct versions of the biosensor, each utilizing a different reporter protein: a fluorescent biosensor (Mer-RFP) and a colorimetric biosensor (Mer-Blue). Mer-RFP enabled real-time monitoring of the culture's response to mercury samples using a plate reader, whereas Mer-Blue was analysed for colour accumulation at the endpoint using a specially designed, low-cost camera setup for harvested cell pellets. Both biosensors exhibited negligible baseline expression of their respective reporter proteins and responded specifically to HgBr2 in pure water. Mer-RFP demonstrated a linear detection range from 1 nM to 1 µM, whereas Mer-Blue showed a linear range from 2 nM to 125 nM. Our biosensors successfully detected a high concentration of ionic mercury in the reaction bucket where artisanal miners produce a mercury-gold amalgam. However, they did not detect ionic mercury in the water from active mining ponds, indicating a concentration lower than 3.2 nM Hg2+-a result consistent with chemical analysis quantitation. Furthermore, we discuss the potential of Mer-Blue as a practical and affordable monitoring tool, highlighting its stability, reliance on simple visual colorimetry, and the possibility of sensitivity expansion to organic mercury.


Asunto(s)
Técnicas Biosensibles , Monitoreo del Ambiente , Mercurio , Mercurio/análisis , Monitoreo del Ambiente/métodos , Colorimetría , Contaminantes Químicos del Agua/análisis , Límite de Detección , Oro/química
2.
Arch Insect Biochem Physiol ; 112(1): e21977, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36254855

RESUMEN

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.


Asunto(s)
Aedes , Femenino , Animales , Aedes/genética , Aedes/metabolismo , Espectrometría de Masas en Tándem , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Larva , Proteínas Recombinantes/metabolismo , ADN Complementario/genética , Clonación Molecular , Hormonas Juveniles/metabolismo
3.
Biomolecules ; 12(7)2022 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-35883546

RESUMEN

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH's preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.


Asunto(s)
Drosophila melanogaster , Hormonas Juveniles , Animales , Clonación Molecular , Drosophila melanogaster/metabolismo , Epóxido Hidrolasas/química , Femenino , Hormonas Juveniles/metabolismo
4.
NMR Biomed ; 34(5): e4169, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-31518036

RESUMEN

31 P MR spectroscopic imaging (MRSI) is a versatile technique to study phospholipid precursors and energy metabolism in the healthy and diseased human brain. However, mainly due to its low sensitivity, 31 P MRSI is currently limited to research purposes. To obtain 3D 31 P MRSI spectra with improved signal-to-noise ratio on clinical 3 T MR systems, we used a coil combination consisting of a dual-tuned birdcage transmit coil and a 31 P eight-channel phased-array receive insert. To further increase resolution and sensitivity we applied WALTZ4 1 H decoupling and continuous wave nuclear Overhauser effect (NOE) enhancement and acquired high-quality MRSI spectra with nominal voxel volumes of ~ 17.6 cm3 (effective voxel volume ~ 51 cm3 ) in a clinically relevant measurement time of ~ 13 minutes, without exceeding SAR limits. Steady-state NOE enhancements ranged from 15 ± 9% (γ-ATP) and 33 ± 3% (phosphocreatine) to 48 ± 11% (phosphoethanolamine). Because of these improvements, we resolved and detected all 31 P signals of metabolites that have also been reported for ultrahigh field strengths, including resonances for NAD+ , NADH and extracellular inorganic phosphate. T1 times of extracellular inorganic phosphate were longer than for intracellular inorganic phosphate (3.8 ± 1.4s vs 1.8 ± 0.65 seconds). A comparison of measured T1 relaxation times and NOE enhancements at 3 T with published values between 1.5 and 9.4 T indicates that T1 relaxation of 31 P metabolite spins in the human brain is dominated by dipolar relaxation for this field strength range. Even although intrinsic sensitivity is higher at ultrahigh fields, we demonstrate that at a clinical field strength of 3 T, similar 31 P MRSI information content can be obtained using a sophisticated coil design combined with 1 H decoupling and NOE enhancement.


Asunto(s)
Encéfalo/diagnóstico por imagen , Espectroscopía de Resonancia Magnética , NAD/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Femenino , Humanos , Masculino , Metaboloma , Fosfatos/análisis , Fosfocreatina/análogos & derivados , Fosfocreatina/metabolismo , Fósforo , Espectroscopía de Protones por Resonancia Magnética , Procesamiento de Señales Asistido por Computador , Factores de Tiempo
5.
Cancer Metab ; 7: 4, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31139406

RESUMEN

BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D-2-hydroxyglutarate (D-2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH-mutated (IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D-2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy. METHODS: We performed 13C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D-2-HG production, cell proliferation rates, and sensitivity to radiotherapy. RESULTS: Significant amounts of 13C from glutamate accumulate in D-2-HG in HCT116-IDH1 wt/R132H but not in HCT116-IDH1 wt/wt . Preventing glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in reduction of D-2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1mut inhibitor AGI-5198. CONCLUSIONS: This work shows that glutamate can be directly processed into D-2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers.

6.
ACS Chem Neurosci ; 10(5): 2591-2601, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30873831

RESUMEN

Imaging of hyperpolarized 13C-labeled substrates has emerged as an important magnetic resonance (MR) technique to study metabolic pathways in real time in vivo. Even though this technique has found its way to clinical trials, in vivo dynamic nuclear polarization is still mostly applied in preclinical models. Its tremendous increase in signal-to-noise ratio (SNR) overcomes the intrinsically low MR sensitivity of the 13C nucleus and allows real-time metabolic imaging in small structures like the mouse brain. However, applications in brain research are limited as delivery of hyperpolarized compounds is restrained by the blood-brain barrier (BBB). A local noninvasive disruption of the BBB could facilitate delivery of hyperpolarized substrates and create opportunities to study metabolic pathways in the brain that are generally not within reach. In this work, we designed a setup to apply BBB disruption in the mouse brain by MR-guided focused ultrasound (FUS) prior to MR imaging of 13C-enriched hyperpolarized [1-13C]-pyruvate and its conversion to [1-13C]-lactate. To overcome partial volume issues, we optimized a fast multigradient-echo imaging method (temporal resolution of 2.4 s) with an in-plane spatial resolution of 1.6 × 1.6 mm2, without the need of processing large amounts of spectroscopic data. We demonstrated the feasibility to apply 13C imaging in less than 1 h after FUS treatment and showed a locally disrupted BBB during the time window of the whole experiment. From detected hyperpolarized pyruvate and lactate signals in both FUS-treated and untreated mice, we conclude that even at high spatial resolution, signals from the blood compartment dominate in the 13C images, leaving the interpretation of hyperpolarized signals in the mouse brain challenging.


Asunto(s)
Barrera Hematoencefálica/fisiología , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Ondas Ultrasónicas , Animales , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Imagen por Resonancia Magnética Intervencional/métodos , Redes y Vías Metabólicas/fisiología , Ratones , Microscopía de Polarización/métodos
7.
Magn Reson Med ; 82(2): 825-832, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30900352

RESUMEN

PURPOSE: To demonstrate a 1 H/31 P whole human brain volume coil configuration for 3 Tesla with separate 31 P transmit and receive components that maintains 1 H MRS performance and delivers optimal 31 P MRSI with 1 H decoupling. METHODS: We developed an 8-channel 31 P receive array coil covering the head to be used as an insert for a commercial double-tuned 1 H/31 P birdcage transmit-receive coil. This retains the possibility of using low-power rectangular pulses for 1 H-decoupled 3D 31 P MRSI (nominal resolution 17.6 cm3 ; acquisition duration 13 min) but increases the SNR with the receive sensitivity of 31 P surface coils. The performance of the combined coil setup was evaluated by measuring 1 H and 31 P SNR with and without the 31 P receive array and by assessing the effect of the receive array on the transmit efficiencies of the birdcage coil. RESULTS: Compared to the birdcage coil alone, the 31 P insert in combination with the birdcage achieved an average 31 P SNR gain of 1.4 ± 0.4 in a center partition of the brain. The insert did not cause losses in 1 H MRS performance and transmit efficiency, whereas for 31 P approximately 20% more power was needed to achieve the same γB1. CONCLUSION: The new coil configuration allows 1 H MRSI and optimal 1 H-decoupled 3D 31 P MRSI, with increased SNR of the human brain without patient repositioning, for clinical and research purposes at 3 Tesla.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Adulto , Algoritmos , Encéfalo/diagnóstico por imagen , Diseño de Equipo , Femenino , Humanos , Masculino , Fantasmas de Imagen
8.
FASEB J ; 33(1): 557-571, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30001166

RESUMEN

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.


Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Ácido Glutámico/metabolismo , Isocitrato Deshidrogenasa/genética , Ácido Láctico/metabolismo , Mutación , Estrés Fisiológico , 4-Aminobutirato Transaminasa/genética , 4-Aminobutirato Transaminasa/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Glutaminasa/genética , Glutaminasa/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismo , Transcriptoma , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Analyst ; 138(4): 1011-4, 2013 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-23304699

RESUMEN

We combined the high MR signal enhancement achieved using dissolution dynamic nuclear polarization (DNP) with a pulsed gradient double spin echo diffusion MR sequence to rapidly and accurately measure the diffusion coefficients of various hyperpolarized (13)C molecules in solution. Furthermore, with a diffusion-weighted imaging sequence we generate diffusion coefficient maps of multiple hyperpolarized metabolites simultaneously. While hyperpolarized experiments can measure rapid, non-equilibrium processes by avoiding signal averaging, continuous signal loss due to longitudinal relaxation (T(1)) complicates quantitation. By correcting for this signal loss, we demonstrate the feasibility of using hyperpolarized (13)C diffusion-weighted MR to accurately measure real-time (seconds) molecular transport phenomena. Potential applications include rapidly measuring molecular binding, cellular membrane transport, in vivo metabolite distribution and establishing a magnetic field independent hyperpolarized parameter.


Asunto(s)
Isótopos de Carbono/química , Imagen de Difusión por Resonancia Magnética/métodos , Soluciones/química
10.
Nature ; 469(7329): 207-11, 2011 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-21150900

RESUMEN

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.


Asunto(s)
Bioingeniería , Lógica , Modelos Biológicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Biología de Sistemas/métodos , Candida albicans , Compartimento Celular , Recuento de Colonia Microbiana , Doxiciclina/farmacología , Estradiol/farmacología , Galactosa/farmacología , Factor de Apareamiento , Péptidos/metabolismo , Péptidos/farmacología , Feromonas/metabolismo , Feromonas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Cloruro de Sodio/farmacología
11.
J Microbiol Biotechnol ; 19(7): 698-708, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19652518

RESUMEN

High rate sulfate reduction under acidic conditions opens possibilities for new process flow sheets that allow the selective recovery of metals from mining and metallurgical waste and process water. However, knowledge about high-rate sulfate reduction under acidic conditions is limited. This paper investigates sulfate reduction in a membrane bioreactor at a controlled pH of 5. Sulfate and formate were dosed using a pH-auxostat system while formate was converted into hydrogen, which was used for sulfate reduction. Sulfide was removed from the gas phase to prevent sulfide inhibition. This study shows a high-rate sulfate-reducing bioreactor system for the first time at pH 5, with a volumetric activity of 188mmol SO(4)(2-)/I/d and a specific activity of 81mmol SO(4)(2-) volatile suspended. The microbial community at the end of the reactor run consisted of a diverse mixed population including sulfate-reducing bacteria.


Asunto(s)
Reactores Biológicos , Sulfatos/química , Bacterias Reductoras del Azufre/aislamiento & purificación , Biodegradación Ambiental , Biomasa , ADN Bacteriano/análisis , ADN Bacteriano/genética , Formiatos/química , Formiatos/metabolismo , Concentración de Iones de Hidrógeno , Residuos Industriales , Membranas Artificiales , Oxidación-Reducción , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , Proteobacteria/metabolismo , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Sulfatos/metabolismo , Sulfuros/química , Sulfuros/metabolismo , Bacterias Reductoras del Azufre/genética , Bacterias Reductoras del Azufre/metabolismo
12.
Sci Signal ; 2(63): ra13, 2009 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-19318625

RESUMEN

Appropriate regulation of the Hog1 mitogen-activated protein kinase (MAPK) pathway is essential for cells to survive osmotic stress. Here, we show that the two sensing mechanisms upstream of Hog1 display different signaling properties. The Sho1 branch is an inducible nonbasal system, whereas the Sln1 branch shows high basal signaling that is restricted by a MAPK-mediated feedback mechanism. A two-dimensional mathematical model of the Snl1 branch, including high basal signaling and a Hog1-regulated negative feedback, shows that a system with basal signaling exhibits higher efficiency, with faster response times and higher sensitivity to variations in external signals, than would systems without basal signaling. Analysis of two other yeast MAPK pathways, the Fus3 and Kss1 signaling pathways, indicates that high intrinsic basal signaling may be a general property of MAPK pathways allowing rapid and sensitive responses to environmental changes.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Equilibrio Hidroelectrolítico/fisiología , Western Blotting , Citometría de Flujo , Chaperonas Moleculares , Levaduras
13.
J Shoulder Elbow Surg ; 18(2): 283-7, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19101177

RESUMEN

HYPOTHESIS: The purpose of this study is to report the functional outcome of the repair of a distal biceps tendon rupture by the use of the endobutton technique. We hypothesized that the endobutton provides excellent strength and clinical results after repair of distal biceps tendon rupture. MATERIALS AND METHODS: Twenty-six patients underwent repair of biceps tendon ruptures by use of an endobutton for fixation of the biceps tendon stump to the radial tuberosity. There were 20 men and three women. The mean age was 52 years (39-75). The dominant side was involved in 11 patients. A partial rupture of the biceps tendon was found in four patients. The average delay in diagnosis was 16 days, with four patients presenting at six weeks or more after trauma. RESULTS: At an average follow-up of 16 months (6-48), 23 of 26 patients were available for follow-up and were examined clinically, radiologically, and by isokinetic testing. The average postoperative Mayo Elbow Performance Score (MEPS) was 94 points. The average Visual Analogue Scale (VAS) for pain was 1.5. Patients regained an almost full range of motion. Average flexion strength recovery was 80% and corresponding recovery of supination strength was 91%. Two patients developed asymptomatic heterotopic ossification seen on standard radiographs. In three patients, the endobutton had apparently disengaged without important difference in functional outcome. In one case, the endobutton had to be removed. There were no neurological complications. CONCLUSION: This study shows that a distal biceps tendon can be safely reattached to the radius by using the endobutton technique, yielding excellent and reproducible results. LEVEL OF EVIDENCE: Level 4; Retrospective case series, no control group.


Asunto(s)
Articulación del Codo , Traumatismos de los Tendones/cirugía , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Ortopédicos/métodos , Dimensión del Dolor , Radiografía , Rango del Movimiento Articular , Estudios Retrospectivos , Rotura , Traumatismos de los Tendones/diagnóstico por imagen
14.
Water Res ; 42(10-11): 2439-48, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18377946

RESUMEN

Many industrial waste and process waters contain high concentrations of sulfate, which can be removed by sulfate-reducing bacteria (SRB). This paper reports on mesophilic (30 degrees C) sulfate reduction at pH 6 with formate as electron donor in a membrane bioreactor with a pH-auxostat dosing system. A mixed microbial community from full-scale industrial wastewater treatment bioreactors operated at pH 7 was used as inoculum. The pH-auxostat enabled the bacteria to convert sulfate at a volumetric activity of 302 mmol sulfate reduced per liter per day and a specific activity of 110 mmol sulfate reduced per gram volatile suspended solids per day. Biomass grew in 15 days from 0.2 to 4 g volatile suspended solids per liter. This study shows that it is possible to reduce sulfate at pH 6 with formate as electron donor at a high volumetric and specific activity with inocula from full-scale industrial wastewater treatment bioreactors operated at neutral pH. The combination of a membrane bioreactor and a pH-auxostat is a useful research tool to study processes with unknown growth rates at maximum activities.


Asunto(s)
Reactores Biológicos , Formiatos/metabolismo , Membranas Artificiales , Sulfatos/metabolismo , Biomasa , Medios de Cultivo , Electrones , Gases , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Tamaño de la Partícula , Aguas del Alcantarillado , Termodinámica
15.
Trends Biochem Sci ; 32(12): 547-54, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17983752

RESUMEN

One major class of G proteins typically functions as heterotrimeric complexes consisting of Galpha, Gbeta and Ggamma subunits. However, recent work in yeast has identified an atypical Galpha protein, Gpa2p, which functions without cognate Gbetagamma subunits. Two novel kelch repeat protein binding partners of Gpa2p, Krh1p and Krh2p, do not function as alternative Gbeta subunits, as initially thought, but rather as Gpa2p effectors. They directly link Gpa2p to protein kinase A, thus forming an adenylate cyclase bypass pathway that enables inputs other than cellular cAMP concentration to affect protein kinase A activity. Because mammalian protein kinase A expressed in yeast is also subject to control by the same bypass pathway, it is exciting to postulate that a functionally similar mechanism might exist in mammalian cells, and that other Galpha proteins could exhibit similar characteristics to Gpa2p.


Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Secuencias Repetitivas de Aminoácido , Adenilil Ciclasas/química , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas de Unión al GTP/química , Transducción de Señal
16.
Proc Natl Acad Sci U S A ; 103(35): 13034-9, 2006 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-16924114

RESUMEN

The cAMP-PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the G(alpha) protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as G(beta) mimics for Gpa2, based on their predicted seven-bladed beta-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved.


Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Dominio Catalítico , AMP Cíclico/metabolismo , Regulación hacia Abajo/genética , Guanosina Trifosfato/metabolismo , Holoenzimas/metabolismo , Ratones , Modelos Biológicos , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo
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