The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.     

Analysis of some tryptophan and phenylalanine metabolites in urine by a straight-phase high-performance liquid chromatographic technique

A high-performance liquid column chromatographic technique is reported for the analysis of some tryptophan and phenylalanine acid metabolites in the urine. An acidified and NaCl-saturated urine sample is loaded on to a C₁₅-bonded silica microcolumn. After washing the microcolumn with clean and deionized water, the metabolites of interest are selectively extracted by successive elutions with organic solvents of variable polarity. Acids are eluted first and the neutral compounds with the next fraction. Basic compounds and other neutral substances of higher polarities were eliminated during the washing procedure.The chromatography was performed in the straight-phase isocratic elution mode utilizing 5-μm silica-gel columns loaded with a triethanolammonium perchlorate—perchloric acid aqueous solution. The separations achieved have permitted the application of the chromatographic technique to the analysis of urinary metabolites with acceptable accuracy.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Contributo alla conoscenza del contingente nervoso corneale

Riassunto Nella cornea del ratto, in tutta quella parte del parenchima che è al margine con la sclerotica, esistono due cospicue formazioni a plesso, una vascolare e l'altra nervosa, in stretto reciproco rapporto topografico e verosimilmente anche funzionale.Il plesso nervoso è formato da fibre di varia provenienza, mieliniche ed amieliniche, fra loro mescolate ed anastomizzate, soprattutto mediante la rete diffusa alla quale danno origine.È molto probabile che a queste due formazioni, vascolare e nervosa, non sia riservato il solo compito di assicurare la nutrizione della cornea; ma penso che esse non siano estranee al meccanismo regolatore dell'umore acqueo.

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Zusammenfassung Der Verfasser hat mit der Bielschowsky-Grosschen Methode und vor allem mit der Schwarzreaktion von Golgi in Epymis norv. die angehäufte Zahl von Nerven studiert, die im Zusammenhang mit dem Hornhautrand stehen und von denen weder die Topographie noch ihre Bedeutung sonderlich bekannt war. Indem der Verfasser diese Methoden mit der Injektion des Blutgefäßsystems verband, hat er festgestellt, daß der plexiforme Nervenapparat enge topographische und funktionelle Beziehungen zu dem sich ringförmig in der gleichen Zone erstreckenden Gefäßplexus hat.Der Verfasser legt außerdem klar, daß dieser vasculonervöse Plexus zur Hornhaut und nicht zu anderen Bildungen gehört. Er schließt ferner, daß diesem angesichts seiner großen Entwicklung eine bemerkenswertere Aufgabe zukommt als jene, lediglich die Ernährung der Hornhaut zu sichern und daß er ein Teil des sinnreichen Regulationsmechanismus des Kammerwassers sei.

     

Structural studies on the murine granulocyte colony-stimulating factor

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein hemopoietic growth factor which regulates the production of granulocytes and macrophages. Reversed-phase microbore high-performance liquid chromatography was employed to purify a number of tryptic and Staphylococcus aureus V8 proteinase peptides generated from approximately 400 pmol G-CSF purified from medium conditioned by lungs from mice previously injected with endotoxin. N-Terminal amino-acid sequence analyses were performed on the parent polypeptide and on four tryptic peptides and one Staphylococcus aureus V8 protease peptide, yielding 68 unique amino-acid assignments; this corresponds to approximately 38% of the molecule.     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)