Processing of influenza HA protein in MDCK cells: Components with different mobilities in polyacrylamide gel electrophoresis and their precursor-product relationships

Summary In influenza virus-infected MDCK cells labelled with¹⁴C-chlorella hydrolysate or³⁵S-methionine a virus-specific protein component is revealed migrating slightly faster than HA protein in polyacrylamide gel electrophoresis. Under chase conditions the component disappears either completely or partially, with a concomitant intensification of the HA band. The rate and extent of this transition are strain-dependent. Both the HA band and the faster moving component are not revealed if the cells are labelled in the presence of 20mM of D-glucosamine. In primary cell cultures of chick embryos a single HA band with a mobility similar to that of the faster moving component in MDCK cells has been observed. It is suggested that the transition of the label from the faster moving component to the HA band reflects the final step of HA processing specific for MDCK cells.With 7 Figures     

Muscle-epidermis interactions affect exoskeleton patterning in Caenorhabditis elegans.

The C. elegans hypodermis is a single epithelial cell layer separated from the musculature by a thin basement membrane on its basal surface. The hypodermis secretes the extracellular material of the cuticle from its apical surface. The regulation of cuticle synthesis and apical secretion is not well understood. UNC-95 is a component of the muscle dense bodies and M-lines, which are integrin-based adhesion complexes required for force transduction to the cuticle. Using gene expression profiling and in vivo assays, we show that, in unc-95 mutant worms, there is an increase in expression levels of a group of hypodermal and pharyngeal genes related to cuticle structure and molting. Moreover, the cuticle structure of unc-95 mutant adult is impaired. Our findings suggest that aberrant force transduction from the structurally impaired muscle attachments across the basement membrane to the underlying hypodermis elicits intercellular signaling that plays a role in regulating cuticle synthesis and patterning.     

Species-Specific Differences in Organization of Orthopoxvirus Kelch-Like Proteins

Organization of orthopoxvirus proteins of the kelch superfamily and their genes were analyzed and compared. Complete genomic sequences of variola (VAR), monkeypox (MPV), vaccinia (VAC), and species-specific regions of cowpox (CPV) viruses were used in the work. Despite the multiplicity of kelch-like proteins in orthopoxviruses, their function is still vague. It has been discovered that the genes of orthopoxvirus kelch-like proteins are localized only to the terminal variable regions of the genome and display species-specific differences in the lengths of the proteins they potentially encode. All the genes belonging to kelch superfamily in the genome of VAR, which has the only host–the man, are mutationally destroyed. However, CPV, displaying the widest host range among orthopoxviruses, encode the most numerous set of kelch-like proteins. Weak homologies between kelch-like proteins of one virus were demonstrated as well as high homologies between isologues of different orthopoxvirus species. The comparison performed suggest that CPV virus is most ancient and may be considered as the ancestor of other orthopoxviruses pathogenic for humans.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Kinetics and mechanism of formation and properties of polymers composed of paired macromolecules

The influence of formation conditions on structure and properties of reaction products from two different macromolecules (paired polymers) such as polystyrene and poly(1,1,2-trichlorobutadiene) or polystyrene and poly(vinyl chloride) is investigated. Mechanical properties, molecular mobility, heat resistance, thermostability, and fire resistance are shown to be regulated over a wide range by changing the molecular weight of the initial polymers, their ratio in the reaction mixture, etc. The interaction of different macromolecules in solution to form paired polymers is analyzed theoretically and experimentally. An analysis of structure and properties of the resulting products by refractometry, viscometry, sedimentation velocity, statistical analysis, and others shows that paired polymers are systems of the “coil-in-coil” type held together by chemical bonds in the zones of mutual penetration.     

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.     

Intracellular cytosolic free calcium concentration in the macula densa and in ascending limb cells at different luminal concentrations of sodium chloride and with added furosemide

The juxtaglomerular apparatus fulfils several important regulatory functions in the kidney, such as tubuloglomerular feedback (TGF) control and control of renin release. The macula densa (MD) cells sense the fluid load by perceiving the distal NaCl concentration via a Na-K-2Cl cotransport system in the luminal cell membrane. It has been proposed that macula densa cell activation may involve changes in intracellular cytosolic free calcium concentration ([Ca2+]i), as one link in the chain of events activating TGF or releasing renin. We therefore investigated the changes in the intracellular calcium concentrations with fura-2, using a video system, in macula densa cells, and compared them with the changes in the corresponding concentrations in the ascending limb of the loop of Henle (c-TAL). The results show that our technique for analysing intracellular cytosolic free calcium in isolated perfused tubules is valid for this purpose, and the Kd value obtained was similar to that found by Grynkiewicz et al. (1985). The intracellular cytosolic free calcium concentration was about 90 nM both in the macula densa and c-TAL cells, and the macula densa cell intracellular cytosolic free calcium concentration increased by about 20 nM when the tubular lumen was perfused with Na and Cl at low concentrations. No significant changes were noted when furosemide was added to the perfusion solutions. We consider it hardly likely that this small change in intracellular cytosolic free calcium concentration can be entirely responsible for full activation of renin release or full inactivation of the TGF control mechanism. It would seem that the signal transmission from the macula densa cells could occur by other routes than through activation of intracellular cytosolic free calcium concentration.     

Single dipole localization: Some numerical aspects and a practical rejection criterion for the fitted parameters

Summary There have been a number of attempts in the last years to localize the generators of brain electromagnetic activity, considering one current dipole as the source model. Single Dipole Localization (SDL) requires the selection of an optimization algorithm (OA). General aspects related with the selection, implementation and evaluation of some of the OA employed for SDL are discussed in this paper. Specifically the performance of two algorithms, those of Hooke-Jeeves and Levenberg-Marquardt, are tested by simulations. Suggestions for including restrictions to the dipole position and comments about some commonly used measures of the goodness of fit are given. Examples of erroneous implementations of these algorithms are also illustrated. A simple graphic rejection criterion, which can be easily used by inexperienced researchers, is introduced and tested in noisy and noise free simulations.The authors are grateful to Roberto D. Pascual Marqui for programming the Hooke-Jeeves algorithm.     

Optimedin: a novel olfactomedin-related protein that interacts with myocilin

Mutations in the MYOC gene may lead to juvenile open-angle glaucoma with high intraocular pressure, and are detected in about 4% of people with adult onset glaucoma. Most of these mutations are found in the third exon of the gene encoding the olfactomedin-like domain located at the C terminus of the protein. Another olfactomedin-related protein, known as noelin or pancortin, is involved in the generation of neural crest cells. Here we describe the identification of a novel olfactomedin-related gene, named optimedin, located on chromosome 1p21 in humans. Optimedin and noelin are both expressed in brain and retina. However, unlike noelin, rat optimedin is also highly expressed in the epithelial cells of the iris and the ciliary body in close proximity to the sites of Myoc expression. In the human eye, optimedin is expressed in the retina and the trabecular meshwork. Both optimedin and myocilin are localized in Golgi and are secreted proteins. The presence of mutant myocilin interferes with secretion of optimedin in transfected cells. Optimedin and myocilin interact with each other in vitro as judged by the GST pulldown, co-immunoprecipitation and far-western binding assays. The C-terminal olfactomedin domains are essential for interaction between optimedin and myocilin, while the N-terminal domains of both proteins are involved in the formation of protein homodimers. We suggest that optimedin may be a candidate gene for disorders involving the anterior segment of the eye and the retina.