Association of fibroblastoid features with the invasivephenotype in human bronchial cancer cell lines

The acquisition of a metastatic phenotype by epithelial cells implicates a series of changes altering their differentiation, their overall behavior and morphology. In the present study, we have examined the relationships between the cellular morphology, E-cadherin expression, matrix metalloproteinases expression and in vitro invasive properties in two human bronchial immortalized cell lines. The (16HBE14o-) cell line which did not show any invasive abilities in the Boyden chamber assay displayed a typical epithelial morphology in monolayer, expressed high levels of E-cadherin and synthesized neither MMP-2 and MT1-MMP nor vimentin. In contrast, the BZR cell line which was highly invasive displayed a more elongated phenotype in monolayer, did not produce E-cadherin but expressed vimentin, MMP-2 and MT1-MMP. Our data therefore suggest that the metastatic progression of broncho-pulmonary cancer cells results in a cellular dedifferentiation and the gain of some mesenchymal attributes (loss of E-cadherin and expression of vimentin) associated with enhanced degradative properties (expression of metalloproteinases).© Rapid Science 1998     

Functional rescue of defective mutant connexons by pairing with wild-type connexons

We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.     

Plasmareninaktivität nach Orthostase und nach Diazoxid bei primärer und renaler Hypertonie

Zusammenfassung 1. Nach Blutdrucksenkung durch schnelle intravenöse Injektion vonDiazoxid kommt es bei 35% der Kranken mit primärem Hochdruck zu einem Anstieg der Plasmareninaktivität. Das Ausbleiben des Reninanstiegs bei der Mehrzahl der fortgeschrittenen primären Hypertonie wird, ebenso wie die abgeschwächte Stimulierbarkeit der Reninsekretion durch Natriummangel, auf eine (adaptiv) verminderte Empfindlichkeit der intrarenalen Receptoren zurückgeführt. Die Blutdrucksenkung durch Diazoxid ist bei positiv und negativ reagierenden Kranken gleich groß.2. Nach einer definiertenOrthostase von 60 min Dauer bei 70° Schräglagerung fehlt ein Plasmareninanstieg bei 63% der untersuchten Patienten mit primärer Hypertonie, wofür ebenfalls eine herabgesetzte Ansprechbarkeit intrarenaler Receptoren verantwortlich gemacht werden muß. Eine Orthostase-negative Reaktion wird ebenso wie eine solche nach Diazoxid häufiger bei primärer Hypertonie in einem fortgeschrittenem, aber auch schon in einem frühen Stadium beobachtet, in dem der Plasmareninanstieg auf Natriumentzug noch intakt ist.3. Die Reninsekretion verhält sich beiprimärer Hypertonie nach Diazoxid, Orthostase und Natriumentzug nicht übereinstimmend, was für die Existenz verschiedener intrarenaler Mechanismen spricht, die die Reninfreisetzung im iuxtaglomerulären Apparat beeinflussen. Patinten mitrenalem Hochdruck weisen dagegen ein übereinstimmendes positives oder negatives Verhalten der Reninsekretion nach Diazoxid und nach Orthostase auf.4. Für diedifferentialdiagnostische Abgrenzung einer funktionell wirksamen Nierenarterienstenose von einer primären Hypertonie ist weder der Diazoxidtest noch — ohne gleichzeitigen Natriumentzug — in der durchgeführten Versuchsanordnung der Orthostasetest geeignet. Zum Nachweis einer funktionell wirksamen Nierenarterienstenose empfiehlt sich die getrennte Plasmareninbestimmung in den Nierenvenen oder als Suchmethode die Doppelstimulation durch 3tägigen Natriumentzug und Orthostase am letzten Tag der Natriumrestriktion.

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Summary 1. After blood pressure reduction by rapid intravenous injection of diazoxide plasma renin activity is increased in 35% of primary hypertension. The absence of plasma renin increase in the majority of advanced primary hypertension is explained by an (adaptive) diminished sensibility of the intrarenal receptors. The reduction of blood pressure is equal in diazoxide responsive and unresponsive patients.2. A plasma renin increase failed in 63% of primary hypertension after upright posture by tilting to 70° for 60 minutes. This result is also explained by a diminished sensibility of the intrarenal receptors for renin secretion. An unresponsive reaction to upright posture and diazoxide is more frequent in advanced primary hypertension, but is observed also in an early stage, in which plasma renin stimulation by sodium deficiency is maintained.3. The stimulation of the renin secretion in primary hypertension is not equal after upright posture, diazoxide and sodium restriction supporting the existence of several intrarenal mechanism controlling the iuxtaglomerular apparatus. In renal hypertension on the contrary, there is a corresponding positive or negative reaction of renin secretion after diazoxide and upright posture.4. For the differential diagnosis of a functional renal artery stenosis neither the diazoxide test nor — without sodium restriction — the orthostase test is suitable. For the evidence of the functional significance of a renal artery stenosis the separated estimation of plasma renin activity in both renal veins or as a sreening test the double stimulation by sodium restriction for 3 days and upright posture is recommended.

     

Suppression of local and systemic responses in streptococcal cell wall-induced acute inflammation of the air pouch by cyclosporine A. Comparison with the effects of two anti-inflammatory bis-benzimidazoles.

Injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide into a subcutaneous air pouch causes local outpouring of neutrophils and macrophages and distant hemopoietic proliferation in spleen and bone marrow. Cyclosporine A (CyA) suppressed neutrophil accumulation and all cell lines of hemopoiesis. trans-1,2-Bis(5-amidino-2-benzimidazolyl)ethene (BBE) also interfered with neutrophil exudation, yet reduced only the erythroid component of the hemopoietic process. The ethane analogue of BBE, on the other hand, did not prevent neutrophil emigration, but held down splenic erythropoiesis and myelopoiesis. All three compounds stimulated streptococcus group A cell wall-derived peptidoglycan polysaccharide uptake by pouch macrophages. CyA being the least active, BBE and its ethane analogue also produced a shift of wear-and-tear pigment from large numbers of small splenic macro-phages into small numbers of large macrophages. The pouch model is very useful in the study of anti-inflammatory compounds and has furnished the first evidence of CyA interference with massive neutrophilic infiltration and with hemopoietic signals.     

Uniparental disomy 7 in Silver--Russell syndrome and primordial growth retardation

Maternal uniparental disomy for the entire chromosome 7 hasso far been reported in three patients with intrauterine andpostnatal growth retardation. Two were detected because theywere homozygous for a cystic fibrosis mutation for which onlythe mother was heterozygous, and one because he was homozygousfor a rare COL1A2 mutation. We investigated 35 patients witheither the Silver-Russell syndrome or primordial growth retardationand their parents with PCR markers to search for uniparentaldisomy 7. Four of 35 patients were found to have maternal disomy,including three with isodisomy and one with heterodisomy. Thedata confirm the hypothetical localization of a maternally imprintedgene (or more than one such gene) on chromosome 7. It is suggestedto search for UPD 7 in families with an offspring with sporadicSilver-Russell syndrome or primordial growth retardation.     

An efficient method to generate chromosomal rearrangements by targeted DNA double-strand breaks in Drosophila melanogaster

Homologous recombination (HR) is an indispensable tool to modify the genome of yeast and mammals. More recently HR is also being used for gene targeting in Drosophila. Here we show that HR can be used efficiently to engineer chromosomal rearrangements such as pericentric and paracentric inversions and translocations in Drosophila. Two chromosomal double-strand breaks (DSBs), introduced by the rare-cutting I-SceI endonuclease on two different mobile elements sharing homologous sequences, are sufficient to promote rearrangements at a frequency of 1% to 4%. Such rearrangements, once generated by HR, can be reverted by Cre recombinase. However, Cre-mediated recombination efficiency drops with increasing distance between recombination sites, unlike HR. We therefore speculate that physical constraints on chromosomal movement are modulated during DSB repair, to facilitate the homology search throughout the genome.     

DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

Summary The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)₄, (GTG)₅, (CA)₈, and (TCC)₅, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.     

Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.     

A new procedure for the determination of molar mass distribution from sedimentation equilibrium runs in an analytical ultracentrifuge (AUC), 1. Measurements with interference optics

A new procedure for the determination of the number-, weight- and z-average molar masses M?_n, M?_w and M?_z and a model molar mass distribution W(M) has been developed from measurements of the sedimentation equilibrium. The method is based on the determination of the experimentally measured reduced concentration profile by extrapolation to zero concentration and direct calculation of the molar mass distribution W(M) by nonlinear regression of the integral equation according to the Simplex procedure. Results obtained in different solvents for the well characterized polystyrene NBS 706 with broad distribution are reasonable. Generally, it can be concluded that the suggested procedure is a suitable possibility for the determination of molar mass average values and the molar mass distribution of polymers if the latter is a log-normal, Schulz-Flory or Poisson distribution.