The association between lumbar paraspinal muscle functional cross-sectional area on MRI and regional volumetric bone mineral density measured by quantitative computed tomography.

Osteosarcopenia is a common condition among elderly and postmenopausal female patients. Site-specific bone mineral density is more predictive of bone-related complications. Few studies have investigated muscle-bone associations. Our results demonstrated that in women, significant positive associations between paraspinal muscles FCSA and vBMD exist at different lumbosacral levels. These regional differences should be considered when interpreting bone-muscle associations in the lumbar spine. INTRODUCTION: There is increasing evidence between bone and muscle volume associations. Previous studies have demonstrated comorbidity between osteoporosis and sarcopenia. Recent studies showed that sarcopenic subjects had a fourfold higher risk of concomitant osteoporosis compared to non-sarcopenic individuals. Although site-specific bone mineral density (BMD) assessments were reported to be more predictive of bone-related complications after spinal fusions than BMD assessments in general, there are few studies that have investigated level-specific bone-muscle interactions. The aim of this study is to investigate the associations between muscle functional cross-sectional area (FCSA) on magnetic resonance imaging (MRI) and site-specific quantitative computed tomography (QCT) volumetric bone mineral density (vBMD) in the lumbosacral region among spine surgery patients. METHODS: We retrospectively reviewed a prospective institutional database of posterior lumbar fusion patients. Patients with available MRI undergoing posterior lumbar fusion were included. Muscle measurements and FCSA were conducted and calculated utilizing a manual segmentation and custom-written program at the superior endplate of the L3-L5 vertebrae level. vBMD measurements were performed and calculated utilizing a QCT pro software at L1-L2 levels and bilateral sacral ala. We stratified by sex for all analyses. RESULTS: A total of 105 patients (mean age 61.5 years and 52.4% females) were included. We found that female patients had statistically significant lower muscle FCSA than male patients. After adjusting for age and body mass index (BMI), there were statistically significant positive associations between L1-L2 and S1 vBMD with L3 psoas FCSA as well as sacral ala vBMD with L3 posterior paraspinal and L5 psoas FCSA. These associations were not found in males. CONCLUSIONS: Our results demonstrated that in women, significant positive associations between the psoas and posterior paraspinal muscle FCSA and vBMD exist in different lumbosacral levels, which are independent of age and BMI. These regional differences should be considered when interpreting bone and muscle associations in the lumbar spine.

Mental health capacity building in refugee primary health care settings in Sub-Saharan Africa: impact, challenges and gaps.

BACKGROUND: In 2015, the United Nations High Commissioner for Refugees started a process of mental health capacity building in refugee primary health care settings in seven countries in Sub-Saharan Africa, ultimately aiming to decrease the treatment gap of mental, neurological and substance use (MNS) conditions in these operations. In 2015 and 2016, a specialized non-governmental organization, the War Trauma Foundation, trained 619 staff with the mental health gap action programme (mhGAP) Humanitarian Intervention Guide (HIG), a tool designed to guide clinical decision making in humanitarian settings. METHODS: This paper describes the results of a process evaluation of a real-life implementation project by an external consultant, one and a half years after starting the programme. RESULTS: The mhGAP-HIG capacity building efforts had various effects contributing to the integration of mental health in refugee primary health care. Facility-and community-based staff reported strengthened capacities to deliver mental health and psychosocial support interventions as well as changes in their attitude towards people suffering from MNS conditions. Service delivery and collaboration amongst different intervention levels improved. The scarcity of specialized staff in these settings was a major barrier, hindering the setting-up of supervision mechanisms. CONCLUSION: Mental health training of non-specialized staff in complex humanitarian settings is feasible and can lead to increased competency of providers. However, capacity building is a 'process' and not an 'event' and mhGAP trainings are only one element in a spectrum of activities aimed at integrating mental health into general health care. Regular supervision and continuing on-the-job training are in fact critical to ensure sustainability.

Histoplasmosis in renal transplant patients in an endemic area at a reference hospital in Medellin, Colombia.

BACKGROUND: Histoplasmosis is an uncommon systemic fungal infection, but it is potentially fatal in immunosuppressed populations. In Latin America, which is considered an endemic area for this mycosis, there have been no published reports regarding the incidence, clinical presentation, morbidity, and mortality of histoplasmosis in renal transplant patients. The objective of this study was to describe cases of histoplasmosis in renal transplant patients treated at the Pablo Tobon Uribe Hospital (Medellin, Colombia) between 2006 and 2013. METHODS: This is a descriptive, retrospective study. RESULTS: The incidence of histoplasmosis in our renal transplant population was 1.1%. The ages of the 9 patients (4 men and 5 women) ranged between 27 and 59 years. In 2 of these patients, histoplasmosis appeared during the first year after transplantation. At the time of transplantation, 66% of patients received induction with alemtuzumab; 88% had a prior rejection episode and required increased immunosuppressive medication; 88% had renal graft dysfunction with creatinine levels >1.5 mg/dL; and the primary clinical presentation was disseminated histoplasmosis followed by the pulmonary form of the disease. Diagnoses were performed by histology in 6 patients, blood culture in 2 patients, and antigenuria in 1 patient. Three patients required treatment with amphotericin B for the severity of their infection, and 2 of these patients died before receiving the cumulative dose of amphotericin B. The 7 remaining patients received itraconazole for 12 months and had a successful treatment response. Regarding complications, 2 patients had hemophagocytic syndrome. At the 1-year follow-up appointment, renal function remained stable in all patients, and no patients had acute rejection or required renal replacement therapy. Thus, the overall mortality rate observed was 22.2%. CONCLUSIONS: In this series, histoplasmosis in renal transplant patients presented as an aggressive opportunistic infection with a higher incidence than that previously reported in the literature. The following risk factors have been associated with histoplasmosis: renal graft dysfunction, previous acute rejection, immunosuppression with tacrolimus-mycophenolate, and induction with alemtuzumab. The clinical presentation of histoplasmosis was nonspecific, which complicated disease diagnosis, and the treatment regimens were highly toxic and associated with significant morbidity and mortality rates.

Neuropatía óptica tóxica inducida por Linezolid / Linezolid induced toxic optic neuropathy

El Linezolid es un antimicrobiano sintético que pertenece a un nuevo grupo, elde las oxazolidinonas, aprobado por la Foods and DrugsAdministration (FDA) para su uso en infecciones por microorganismos gram positivos, staphylococcusspp y enterococcusspp, resistentes a la oxacilina y a la vancomicina.Dentro de sus usos principales están enfermedades como neumonías nosocomiales, infecciones de piel y tejidos blandos. Se describe el caso de una paciente con neuropatía óptica asociada al linezolid. La mayoría de estos casos han sido documentados en pacientes que recibieron una terapia superior a los 28 días recomendados.Los médicos debemos estar atentos a diferentes efectos adversos asociados con el linezolid como la neuropatía óptica, aunque es mayor el riesgo de leucopenia, trombocitopenia y neuropatía periférica, asociados a este medicamento (AU)

Linezolid is a new class of synthetic antimicrobial, belonging to the family of oxazolidinones approved by the Food and Drugs Administration for use in gram positive infections by staphylococcus spp and enterococcus spp, resistant tooxacillin and vancomycin. Among its mains uses are diseases such as nosocomial pneumonia, kin infection and soft tissue We describe the case of a patient with optic neuropathy associated with linezolid, most of these cases have been documented in patients who were in therapy at 28 days than recommended, physicians shouldbe alert to various adverse effects associated with linezolid such as optic neuropathy, but there is anincreased risk of leucopenia, thrombocytopenia and peripheral neuropathy associated with this medicine (AU)

RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells.

Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Determinación de la concentración plasmática de acetaminofén después de administrar 1 g cada 4 horas en 4 dosis sucesivas / Plasmatic concentration of acetaminophen after administration of 1 g every 4 hours and doses

PROPOSITO: Determinar la concentración plasmática de acetaminofén después de administrar 1g cada 4 horas en 4 dosis sucesivas. PACIENTE Y METODO: Se seleccionaron 20 pacientes sanos con un promedio de edad de 22 años y un peso promedio de 59 Kg, a quienes se les administró 1g de acetaminofén cada 4 horas en 4 dosis sucesivas. Una hora después de cada dosis se tomó una muestra de sangre mediante venopunción y se cuantificó el nivel plasmático del medicamento usando la tecnología de inmunovaloración de polarización fluorescente. RESULTADOS: La mediana de la concentración plasmática de todos los pacientes después de cada una de las 4 dosis fue de 15.53, 17.27, 16.98 y 23.21 lo cual implicó un aumento del 11.4 entre la primera y la segunda dosis, una disminución del 1.6 entre la segunda y la tercera dosis. DISCUSION: La administración oral de administración oral de acetaminofén en dosis de 500 mg permite alcanzar una concentración máxima que fluctúa entre 6 y 8 mg de plasma. Su administración en dosis de 1g muestra una cinética similar, alcanzando en promedio una concentración máxima de 15ml una hora después de la ingestión y una vida media aproximada de 90 min. CONCLUSION: La concentración plasmática de acetaminofén, una horas después de administrar 4 dosis sucesivas de 1g cada 4 horas, fue en promedio de 15.5, 17.2, 16.9 y 23.2ml respectivamente.

Uso de máscara laríngea en el trasplante renal / Use of the laryngeal mask in the reanl transplantation

Desde su introducción en la práctica de la Anestesiología, la máscara laríngea ha sido utilizada en una gran variedad de procedimientos quirúrgicos, sin embargo no existe literatura que refiera su uso rutinario en pacientes sometidos a transplante renal. En el Hospital Universitario San Vicente de Paúl de Medellín, la máscara laríngea se utiliza éxitosamente en este tipo de procedimientos desde hace mas de cinco años, obteniendo beneficios iguales o mejores a la intubación endotraqueal y con una tasa igual o menor de complicaciones. Este trabajo describe la experiencia del grupo de Anestesiólogos de Servicio de Pensionados de este Hospital con esta clase de procedimientos

Fite stain positivity in Rhodococcus equi: yet another acid-fast organism in respiratory cytology--a case report.

Rhodococcus equi is an aerobic Gram-positive and acid-fast coccobacillus that may cause cavitary pneumonia in immunocompromised hosts such as HIV-infected patients. Numerous Grocott's methenamine silver (GMS)-positive organisms were initially noted on the direct smear; a minor number of acid-fast organisms were seen in the Thin-Prep slide. Since the abundant mucous material with the attached organisms seen in conventional smears may be lost in liquid-based preparations, more sensitive stains such as Fite, as well as a more diligent search for organisms, is needed. This case illustrates the importance of careful selection and evaluation of special stains in sputum specimens.

Vulvar keratinizing squamous cell carcinoma in a 26-year-old woman.

A 26-year-old Hispanic woman complaining of "itching" and "herpetic lesions" on the vulva for 9 months was seen at a university hospital. On physical examination, multiple vulvar masses were noted. Biopsies taken from these lesions showed invasive keratinizing squamous cell carcinoma. The vulvectomy specimen revealed 4 tumor masses, the largest located on the mons pubis. Although the incidence of vulvar intraepithelial neoplasia has increased in recent years, only very few cases of invasive carcinoma have been reported in young women. The tumors that occur at a younger age characteristically have basaloid or warty histology, in contrast to those occurring in older women, which usually are well-differentiated keratinizing carcinomas. We believe this is an unusual case of vulvar squamous cell carcinoma. In addition to our patient's young age, her tumor had a histologic profile usually found in lesions of an elderly woman. The tumor was negative for human papillomavirus by polymerase chain reaction analysis and was positive for p53 by immunohistochemistry.

Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.

Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends.

Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. Our search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein 'receptor' on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining revealed that these structures colocalized extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin became evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removed Arp1 from microtubules but did not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also dispersed the Golgi apparatus and eliminated the microtubule-associated staining pattern. The effect on dynactin was rapidly reversible and, following acetate washout, punctate dynactin was detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, our results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport.

Opposing motor activities of dynein and kinesin determine retention and transport of MHC class II-containing compartments.

MHC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments that are generated in the endosomal pathway. The class II molecules are targetted to early lysosomal structures, termed MIIC, where they interact with antigenic fragments and are subsequently transported to the cell surface. We previously visualised vesicular transport of MHC class II-containing early lysosomes from the microtubule organising centre (MTOC) region towards the cell surface in living cells. Here we show that the MIIC move bidirectionally in a 'stop-and-go' fashion. Overexpression of a motor head-deleted kinesin inhibited MIIC motility, showing that kinesin is the motor that drives its plus end transport towards the cell periphery. Cytoplasmic dynein mediates the return of vesicles to the MTOC area and effectively retains the vesicles at this location, as assessed by inactivation of dynein by overexpression of dynamitin. Our data suggest a retention mechanism that determines the perinuclear accumulation of MIIC, which is the result of dynein activity being superior over kinesin activity. The bidirectional nature of MIIC movement is the result of both kinesin and dynein acting reciprocally on the MIIC during its transport. The motors may be the ultimate targets of regulatory kinases since the protein kinase inhibitor staurosporine induces a massive release of lysosomal vesicles from the MTOC region that is morphologically similar to that observed after inactivation of the dynein motor.

Localization of motor-related proteins and associated complexes to active, but not inactive, centromeres.

Multicentric chromosomes are often found in tumor cells and certain cell lines. How they are generated is not fully understood, though their stability suggests that they are non-functional during chromosome segregation. Growing evidence has implicated microtubule motor proteins in attachment of chromosomes to the mitotic spindle and in chromosome movement. To better understand the molecular basis for the inactivity of centromeres associated with secondary constrictions, we have tested these structures by immunofluorescence microscopy for the presence of motor complexes and associated proteins. We find strong immunoreactivity at the active, but not inactive, centromeres of prometaphase multicentric chromosomes using antibodies to the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1 and p150 Glued ), the kinesin-related proteins CENP-E and MCAK and the proposed structural and checkpoint proteins HZW10, CENP-F and Mad2p. These results offer new insight into the assembly and composition of both primary and secondary constrictions and provide a molecular basis for the apparent inactivity of the latter during chromosome segregation.

Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon.

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761-2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617-633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.

Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

Multiple mouse chromosomal loci for dynein-based motility.

Dyneins are multisubunit mechanochemical enzymes capable of interacting with microtubules to generate force. Axonemal dyneins produce the motive force for ciliary and flagellar beating by inducing sliding between adjacent microtubules within the axoneme. Cytoplasmic dyneins translocate membranous organelles and chromosomes toward the minus ends of cytoplasmic microtubules. Dynactin is an accessory complex implicated in tethering cytoplasmic dynein to membranous organelles and mitotic kinetochores. In the studies described here, we have identified a number of new dynein genes and determined their mouse chromosomal locations by interspecific backcross analysis. We have also mapped several dynein and dynactin genes cloned previously. Our studies provide the first comprehensive attempt to map dynein and dynactin genes in mammals and provide a basis for the further analysis of dynein function in development and disease.

Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization.