RESUMEN
Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon's space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.
Asunto(s)
Colágeno/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Miopía/diagnóstico por imagen , Miopía/terapia , Esclerótica/efectos de los fármacos , Esclerótica/diagnóstico por imagen , Microscopía de Generación del Segundo Armónico/métodos , Animales , Miopía/metabolismo , Conejos , Esclerótica/metabolismoRESUMEN
Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.
Asunto(s)
Microtúbulos/química , Pinzas Ópticas , Óptica y Fotónica/métodos , Citoesqueleto/química , Citoesqueleto/metabolismo , Dineínas/química , Dineínas/aislamiento & purificación , Dineínas/metabolismo , Microscopía/métodos , Microtúbulos/metabolismoRESUMEN
Bacteria have long been ideal model systems for studying many biological phenomena. But when it comes to motility, we are quite often just figuring out the mechanisms underlying their ability to move in liquid or on surfaces. In the last few decades, research has emphasized the importance for bacteria to be able to adhere to and move on surfaces in order to form complex bacterial communities called biofilms. To better understand the multiple chemical and biophysical mechanisms responsible for the initial interactions of bacteria on surfaces that develop into biofilms, we present here low-cost and easy-to-implement protocols to quantitatively analyze the movement of single bacteria on surfaces by microscopy. These protocols are presented in the case of the human pathogen Neisseria gonorrhoeae that moves on surfaces solely powered by Type IV pili, motility referred to as twitching motility. These methods, however, are applicable for any motile bacteria interacting with surfaces. The precise quantification of motility coupled with genetic tools will enable us to precisely dissect the mechanisms and dynamics of bacterial surface motility which are still poorly understood.
Asunto(s)
Neisseria gonorrhoeae/citología , Humanos , Imagenología Tridimensional , Movimiento , Propiedades de SuperficieRESUMEN
A time-to-event model was developed to study the predictive factors of immunosuppressive efficacy in renal transplant patients and to investigate longitudinal calcineurin inhibitor (CNI) and mycophenolic acid (MPA) coexposures and patient characteristics as potential covariates. The efficacy end point included acute rejection (AR), graft loss, and death. Data from 222 patients were analyzed: 23 events were observed in 126 patients receiving cyclosporine as compared with 15 events in 96 patients receiving tacrolimus (P = 0.61) in the first 2 years posttransplantation. Each 1-mg·h/l increase of MPA area under the plasma concentration vs. time curve was associated with a 4% decreased risk of an event (hazard ratio (HR) = 0.96; 95% confidence interval (CI): 0.93-0.99). The onset of cytomegalovirus infection/disease significantly increased this risk (HR = 10.9; 95% CI: 6.5-21.7). Within the observed ranges, CNI exposures were not significantly associated with efficacy (i.e., AR, graft loss, and death). This work advocates the avoidance of unnecessary high CNI dosing and puts forward new arguments for MPA concentration monitoring.
Asunto(s)
Inhibidores de la Calcineurina , Inmunosupresores/uso terapéutico , Trasplante de Riñón , Ácido Micofenólico/uso terapéutico , Ciclosporina/uso terapéutico , Infecciones por Citomegalovirus/prevención & control , Quimioterapia Combinada , Rechazo de Injerto/prevención & control , Humanos , Modelos Logísticos , Modelos Biológicos , Estudios Multicéntricos como Asunto , Ensayos Clínicos Controlados Aleatorios como Asunto , Tacrolimus/uso terapéuticoRESUMEN
Cyclosporin A (CsA) is a substrate for cytochrome P450 3A and the efflux transporter P-glycoprotein (P-gp; ABCB1), both abundantly expressed in the kidney. In a long-term follow-up of a cohort of patients who had received kidney transplants between the years 1990 and 2005, we retrospectively investigated the effect of CYP3A4, CYP3A5, and ABCB1 polymorphisms in kidney graft donors on recipients' renal function and risk of subsequent graft loss. DNA samples from 227 donors and clinical data from the 259 respective recipients were analyzed. Graft loss was significantly associated with the presence of the ABCB1 variant haplotype 1236T/2677T/3435T in the donor (1236T/2677T/3435T vs. other haplotypes: hazard ratio = 9.346; 95% confidence interval (CI) (2.278-38.461); P = 0.0019) and with previous episodes of acute organ rejection (hazard ratio = 3.077; 95% CI (1.213-7.812); P = 0.0178). The variant haplotype was also associated with a greater decrease in renal function (homozygotes for TTT -3.047 mlxmin(-1)/year; heterozygotes for TTT -4.435 mlxmin(-1)/year; others -2.186 mlxmin(-1)/year; P = 0.0240). The study showed that the presence of ABCB1 polymorphisms in donors influences long-term graft outcome adversely with decrease in renal function and graft loss in transplant recipients receiving CsA.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Ciclosporina/uso terapéutico , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/genética , Inmunosupresores/uso terapéutico , Trasplante de Riñón/fisiología , Riñón/fisiología , Polimorfismo Genético/genética , Polimorfismo Genético/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP , Adulto , Anciano , Análisis de Varianza , Estudios de Cohortes , Citocromo P-450 CYP3A/genética , Femenino , Estudios de Seguimiento , Genotipo , Haplotipos , Humanos , Estimación de Kaplan-Meier , Pruebas de Función Renal , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Donantes de Tejidos/estadística & datos numéricos , Adulto JovenRESUMEN
End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Células CHO , Diferenciación Celular/fisiología , Cricetinae , Cricetulus , Dimerización , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/ultraestructura , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.
Asunto(s)
Microtúbulos/metabolismo , Animales , Fenómenos Biomecánicos , Simulación por Computador , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Erizos de MarRESUMEN
The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Schizosaccharomyces , Sistema Libre de Células , Proteínas de Choque Térmico/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Microscopía Fluorescente , Schizosaccharomyces/química , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.
Asunto(s)
Microtúbulos/química , Microtúbulos/metabolismo , Algoritmos , Tampones (Química) , Dimerización , Guanosina Trifosfato/metabolismo , Rayos Láser , Óptica y Fotónica , Estructura Cuaternaria de Proteína , Sensibilidad y Especificidad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Within the period October 1987-September 1990, the authors submit 130 patients to clinical examination and ultrasonography the latter performed by adapting commercially available ultrasonograph type Bruel and Kjaer 3405 and Shimasonic. Among the investigated individuals, 25 exhibited oculo-orbital tumours of various types. The cases are assessed considering the following for establishing the diagnosis: clinical examination, ultrasonography, fluorescein angiography, radiotracer and morphopathological examinations. Besides the use an adapted device, the authors propose a simple and fast morphopathological method: the fixation in formaldehyde solution as long as 48 hours.
Asunto(s)
Neoplasias del Ojo/diagnóstico por imagen , Melanoma/diagnóstico por imagen , Neoplasias Orbitales/diagnóstico por imagen , Retinoblastoma/diagnóstico por imagen , Diagnóstico Diferencial , Neoplasias del Ojo/secundario , Humanos , Neoplasias Orbitales/secundario , UltrasonografíaRESUMEN
The authors present a study made in the period November 1987-1989 on 108 subjects examined clinically echographic and also with some other investigation methods (radiology, laboratory with radio-tracers). There are presented the results obtained on structures of pathology (strokes, primary or metastatic intraocular tumours, medical diseases of the ocular globe). The concordance and disagreement between the results obtained using the investigation with ultrasounds and the clinical investigation completed with some other investigation methods is expressed in the level of fiability of the echographic method (on structures of diseases and generally). The confirmations are justified by: the intraoperative diagnosis, the postoperative evolution, the therapeutical test and also by the histopathological exam in the cases in which this was affected.
Asunto(s)
Oftalmopatías/diagnóstico por imagen , Enfermedades Orbitales/diagnóstico por imagen , Adolescente , Adulto , Diagnóstico Diferencial , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ultrasonografía/instrumentación , Ultrasonografía/métodosRESUMEN
In 1977, coproparazitological examinations, prophylactic and therapeutical studies were carried on 225 children in 14 day nurseries and homes in Cluj-Napoca; 21.57% of children suffered from intestinal parasitosis, giardiasis being found in 92.13% of the infested children. The high incidence of giardiasis led the authors to investigate the hygienic level of the environment. The high frequency of enterobacteriaceae opportunists (77.59%), identified on the hands of the personnel and on certain objects manipulated by the children, pointed to fecal pollution and poor sanitary conditions. The contaminated objects are sources of propagation of Giardia lamblia cysts. The increased incidence of giardiasis and of enterobacteriaceae opportunists in the environment were found both in the communities with many children and in those with few children.