Polarized cells, polar actions.

The recognition of polar bacterial organization is just emerging. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. The significance of this observation remains unclear. Polarity in bacteria poses many problems, including the necessity for a mechanism for asymmetrically distributing proteins as well as a mechanism by which polar localization is maintained. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35). The constraining features for membrane components are not known. For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. In most cases of cellular asymmetry, bacteria are able to discriminate between the new pole and the old pole and to utilize this information for localization specificity. The maturation of new pole to old pole appears to be a common theme as well. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized.     

Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.     

Identification of a yeast artificial chromosome clone spanning a translocation breakpoint at 7q32.1 in a Smith-Lemli-Opitz syndrome patient.

Smith-Lemli-Opitz syndrome (SLOS) is a mental retardation/multiple congenital anomaly syndrome. The gene(s) involved has not been mapped or cloned, but, recently, a biochemical abnormality in cholesterol biosynthesis has been shown to occur in most SLOS patients. The defect is suspected to occur in the penultimate step of the cholesterol pathway, involving the enzyme 7-dehydrocholesterol reductase, which has not been isolated. On the basis of the hypothesis that a de novo balanced translocation [t(7;20)(q32.1;q13.2)] in an SLOS patient directly interrupts the SLOS gene, positional cloning techniques are being employed to localize and identify the SLOS gene. We report the identification of a chromosome 7-specific YAC that spans the translocation breakpoint, as detected by FISH. This is the first study narrowing a candidate SLOS region and placing it on physical and genetic maps of the human genome.     

Opposing effects of human peripheral blood lymphocytes on the growth of cultured leukemia cell lines

Natural killing of two human leukemia cell lines (K562 and Molt 4) in a soft-agar, clonagenic assay was shown to be the result of two opposite yet concurrent processes: target cell colony stimulation and inhibition. The stimulatory effect was demonstrable when the effector lymphocytes and target cells were separated in contiguous agar layers, suggesting mediation by a soluble factor. Similarly, stimulation occurred when the effector lymphocytes and target cells were combined at low effector-target cell ratios that do not favor direct cell contact. Target colony inhibition was found to be dominant when large E:T ratios were employed. Both target-effector binding and natural killing were significantly reduced in medium devoid of divalent cations.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

When similar beginnings lead to different ends: Constraints and diversity in cirripede larval development

Summary

Cirripedes are fascinating models for studying both functional constraints and diversity in larval development. Adult cirripedes display an amazing variation in morphology from sessile suspension feeders that still retain many crustacean characters to parasites that have lost virtually all arthropod traits. In contrast, cirripede larval development follows a common scheme with pelagic larvae comprising a series of nauplii followed by a cyprid. Variations are mostly concerned with whether or not the nauplii are feeding and the degree of abbreviation of development, culminating in species where the larvae hatch as cyprids. The cypris larvae are very similar among the ingroups of the Cirripedia, but interesting variations occur in structures used for substrate location and attachment. The cyprid is specialized to both swim through the water and actively explore the substratum by walking on the antennules and using an array of sensory organs in search for a suitable site to attach. This unique morphology and behavior of the cyprid have enabled the Cirripedia to colonize widely different habitats ranging from hard rock to soft animal tissue. Yet, the cyprid can metamorphose into juveniles as different as a setose feeding barnacle and the vermiform stages of the parasitic forms. This emphasizes the importance of the cyprid as one of the key features for the evolutionary success of the Cirripedia.     

An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non‐intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non‐stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA_i to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than‐average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.     

Mortality,Morbidity and Health-Seeking Behaviour during the Ebola Epidemic 2014–2015 in Monrovia Results from a Mobile Phone Survey

Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0.33/10,000 persons/day (95%CI:0.25–0.43) and an Ebola-specific mortality rate of 0.06/10,000 persons/day (95%-CI:0.03–0.11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.     

Sequence-dependent dynamics in duplex DNA

The submicrosecond bending dynamics of duplex DNA were measured at a single site, using a site-specific electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of the mean squared amplitude of bending relative to the end-to-end vector defined by the weakly bending rod model. The bending dynamics monitored at the single site varied when the length and position of a repeated AT sequence, distant from the spin probe, were changed. As the distance between the probe and the AT sequence was increased, the mean squared amplitude of bending seen by the probe due to that sequence decreased. A model for the sequence-dependent internal flexural motion of duplex DNA, which casts the mean squared bending amplitudes in terms of sequence-dependent bending parameters, has been developed. The best fit of the data to the model occurs when the (AT)(n) basepairs are assumed to be 20% more flexible than the average of the basepairs within the control sequence. These findings provide a quantitative basis for interpreting the kinetics of biological processes that depend on duplex DNA flexibility, such as protein recognition and chromatin packaging.