E pluribus unum: towards a universal mechanism for spindle assembly

Recent data have revealed that the mitotic spindle might form by centrosome-independent mechanisms, even in centrosome-containing cells. This suggests that spindle assembly might proceed by a generally conserved acentrosomal mechanism in all higher eukaryotes, regardless of the presence of centrosomes. Thus, what is the role of centrosomes in mitosis? We propose that these organelles are needed to generate radial arrays of microtubules that integrate preassembled (by centrosome-independent mechanisms) spindle components into a common spindle and orientate the spindle within malleable animal cells.     

Biosynthesized matrix provides a key role for survival signaling in bronchial epithelial cells

The extracellular matrix (ECM) influences a variety of cellular functions, including survival, adhesion molecule expression, differentiation, and migration. The ECM composition of the epithelial basement membrane is altered in asthmatics. In this study, we elucidate the major survival signals received by bronchial epithelial cells in vitro by studying the effects of a variety of ECM factors and soluble growth factors on bronchial epithelial cell survival. Our findings indicate that the insulin family of soluble growth factors provides important survival signals but also that adhesion to ECM is a crucial determinant of bronchial epithelial cell survival. In the BEAS-2B bronchial epithelial cell line, collagens I and IV, laminin, fibronectin, and vitronectin provide significant levels of protection from apoptosis. Tenascin-C has no effect, whereas elastin and collagen V increase apoptosis to above control levels. BEAS-2B cells secrete their own biosynthesized matrix (BSM), which also provides rescue from apoptosis. Protection by collagen I, fibronectin, and vitronectin was found to be via an RGD domain. Laminin-, collagen IV-, and BSM-mediated survival is not RGD dependent. Primary bronchial epithelial cells exhibit a similar pattern of apoptosis rescue to the BEAS-2B cell line, although we did not observe any vitronectin-mediated protection in the primary cells. These data indicate that bronchial epithelial cell survival is dependent both on soluble growth factors and on a variety of ECM-derived signals.     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

Myosin-II-dependent localization and dynamics of F-actin during cytokinesis

BACKGROUND: The assembly of an F-actin- and myosin-II-containing contractile ring (CR) is required for cytokinesis in eukaryotic cells. Interactions between myosin II and actin in the ring are believed to generate the force that constricts the cell into two daughters. The mechanism(s) that contribute to the spatially and temporally regulated assembly and disassembly of the CR at the cell equator are poorly understood. RESULTS: We generated an LLCPK1 epithelial cell line that stably expresses GFP-actin. Live confocal imaging showed accumulation of GFP-actin in the equatorial cortex from late anaphase through cytokinesis. Fluorescence recovery after photobleaching (FRAP) experiments showed that actin in the CR is highly dynamic (t(1/2) = 26 s). In some cells, movement of GFP-actin toward the equatorial region was observed and contributed to FRAP. Blocking actin dynamic turnover with jasplakinolide demonstrates that dynamic actin is required for CR formation and cytokinesis. To test the role of myosin II in actin turnover and transport during CR formation, we inhibited myosin light-chain kinase with ML7 and myosin II ATPase activity with blebbistatin. Inhibition of myosin light-chain phosphorylation resulted in clearance of GFP-actin from the equatorial region, a reduction in myosin II in the furrow, and inhibition of cytokinesis. Treatment with blebbistatin did not block CR formation but reduced FRAP of GFP-actin and prevented completion of cytokinesis. CONCLUSIONS: These results demonstrate that the majority of actin in the CR is highly dynamic and establish novel roles for myosin II in the retention and dynamic turnover of actin in the CR.     

Activated mutant of Galpha(12) enhances the hyperosmotic stress response of NIH3T3 cells

Heterotrimeric G protein G12 stimulates diverse physiological responses including the activities of Na+/H+ exchangers and Jun kinases. We have observed that the expression of the constitutively activated, GTPase-deficient mutant of Galpha(12) (Galpha(12)QL) accelerates the hyperosmotic response of NIH3T3 cells as monitored by the hyperosmotic stress-stimulated activity of JNK1. The accelerated response appears to be partly due to the increased basal activity of JNK since cell lines-such as NIH3T3 cells expressing JNK1-in which JNK activity is elevated, show a similar response. NIH3T3 cells expressing Galpha(12)QL also display heightened sensitivity to hyperosmotic stress. This is in contrast to JNK1-NIH3T3 cells that failed to enhance sensitivity although they do exhibit an accelerated hyperosmotic response. Reasoning that the increased sensitivity seen in Galpha(12)QL cells is due to a signaling component other than JNK, the effect of dimethyamiloride, an inhibitor of Na+/H+ exchanger in this response, was assessed. Treatment of vector control NIH3T3 cells with 50 microM dimethylamiloride potently inhibited their hyperosmotic response whereas the response was only partially inhibited in Galpha(12)QL-NIH3T3 cells. These results, for the first time, identify that NHEs are upstream of the JNK module in the hyperosmotic stress-signaling pathway and that Galpha(12) can enhance this response by modulating either or both of these components namely, JNKs and NHEs in NIH3T3 cells.     

Acute appendicitis and bathrooms in three samples of British children

The occurrence of appendicectomy in three national samples of British children was analysed in relation to household amenities, crowding in the home, and social class. The risk of having the operation depended on the amenities present in the home, in particular whether or not there was a bathroom. This risk was independent of social class.The findings support a relation between acute appendicitis and Western hygiene, which would explain the geographical distribution of the disease and its changing incidence over time. In the developing world, where children grow up in conditions of poor hygiene, there may be outbreaks of appendicitis when housing improves.     

Study of middle ear disease using tympanometry in general practice.

Tympanometry was used to provide evidence of middle ear effusions in a prospective study of middle ear disease in 264 children aged 3 months to 6 years in general practice. Adequate measurements on both ears were obtained in 220 children, of whom 68 (31%) had evidence of middle ear effusion in one ear (29 children) or both ears (39 children) at entry to the study. In 28 (42%) of the 68 children persistence of the tympanometric findings was recorded for at least three months. Children of European descent were more likely to have evidence of middle ear effusion at the initial examination compared with African and West Indian children, as were those children whose siblings had a positive history of otitis media compared with those whose siblings had no such history. Children under 3 years were more likely to have evidence of an effusion than older children. Middle ear effusion as shown by tympanometry was not associated with a previous history of otitis media in the child but was associated with recent symptoms of respiratory infection or otalgia. A previous consultation for otitis media was, however, strongly associated with a greater likelihood of a consultation for otitis media during the follow up period. Comparing evidence of effusion by tympanometry with that by pneumatic otoscopy showed that using the appearance of the eardrum alone the sensitivity of otoscopy was 55%; the addition of mobility improved the sensitivity to 76% with little reduction in specificity. Further studies on populations using tympanometry are needed to determine the natural history, aetiology, and indications for referring children with middle ear effusion.     

Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer''s disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer''s Disease Research Center (ADRC) and Alzheimer''s Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10⁻¹⁰) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.     

Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral symptoms displayed by individuals with autism. While structural neuroimaging findings have provided some insights into brain abnormalities in autism, the consistency of such findings is questionable. Functional neuroimaging, on the other hand, has been more fruitful in this regard because autism is a disorder of dynamic processing and allows examination of communication between cortical networks, which appears to be where the underlying problem occurs in autism. Functional connectivity is defined as the temporal correlation of spatially separate neurological events1. Findings from a number of recent fMRI studies have supported the idea that there is weaker coordination between different parts of the brain that should be working together to accomplish complex social or language problems^2,3,4,5,6. One of the mysteries of autism is the coexistence of deficits in several domains along with relatively intact, sometimes enhanced, abilities. Such complex manifestation of autism calls for a global and comprehensive examination of the disorder at the neural level. A compelling recent account of the brain functioning in autism, the cortical underconnectivity theory,^2,7 provides an integrating framework for the neurobiological bases of autism. The cortical underconnectivity theory of autism suggests that any language, social, or psychological function that is dependent on the integration of multiple brain regions is susceptible to disruption as the processing demand increases. In autism, the underfunctioning of integrative circuitry in the brain may cause widespread underconnectivity. In other words, people with autism may interpret information in a piecemeal fashion at the expense of the whole. Since cortical underconnectivity among brain regions, especially the frontal cortex and more posterior areas ^3,6, has now been relatively well established, we can begin to further understand brain connectivity as a critical component of autism symptomatology.A logical next step in this direction is to examine the anatomical connections that may mediate the functional connections mentioned above. Diffusion Tensor Imaging (DTI) is a relatively novel neuroimaging technique that helps probe the diffusion of water in the brain to infer the integrity of white matter fibers. In this technique, water diffusion in the brain is examined in several directions using diffusion gradients. While functional connectivity provides information about the synchronization of brain activation across different brain areas during a task or during rest, DTI helps in understanding the underlying axonal organization which may facilitate the cross-talk among brain areas. This paper will describe these techniques as valuable tools in understanding the brain in autism and the challenges involved in this line of research. Download video file.^{(73M, mov)}     

Axon response to guidance cues is stimulated by acetylcholine in Caenorhabditis elegans

Gradients of acetylcholine can stimulate growth cone turning when applied to neurons grown in culture, and it has been suggested that acetylcholine could act as a guidance cue. However, the role acetylcholine plays in directing axon migrations in vivo is not clear. Here, we show that acetylcholine positively regulates signaling pathways that mediate axon responses to guidance cues in Caenorhabditis elegans. Mutations that disrupt acetylcholine synthesis, transportation, and secretion affect circumferential axon guidance of the AVM neuron and in these mutants exogenously supplied acetylcholine improves AVM circumferential axon guidance. These effects are not observed for the circumferential guidance of the DD and VD motor neuron axons, which are neighbors of the AVM axon. Circumferential guidance is directed by the UNC-6 (netrin) and SLT-1 (slit) extracellular cues, and exogenously supplied acetylcholine can improve AVM axon guidance in mutants when either UNC-6- or SLT-1-induced signaling is disrupted, but not when both signaling pathways are perturbed. Not in any of the mutants does exogenously supplied acetylcholine improve DD and VD axon guidance. The ability of acetylcholine to enhance AVM axon guidance only in the presence of either UNC-6 or SLT-1 indicates that acetylcholine potentiates UNC-6 and SLT-1 guidance activity, rather than acting itself as a guidance cue. Together, our results show that for specific neurons acetylcholine plays an important role in vivo as a modulator of axon responses to guidance cues.