The interaction between vitamin D receptor polymorphisms and sun exposure around time of diagnosis influences melanoma survival

Evidence on the relationship between the vitamin D pathway and outcomes in melanoma is growing, although it is not always clear. We investigated the impact of measured levels of sun exposure at diagnosis on associations of vitamin D receptor gene (VDR) polymorphisms and melanoma death in 3336 incident primary melanoma cases. Interactions between six SNPs and a common 3′‐end haplotype were significant (p < .05). These SNPs, and a haplotype, had a statistically significant association with survival among subjects exposed to high UVB in multivariable regression models and exerted their effect in the opposite direction among those with low UVB. SNPs rs1544410/BsmI and rs731236/TaqI remained significant after adjustment for multiple testing. These results suggest that the association between VDR and melanoma‐specific survival is modified by sun exposure around diagnosis, and require validation in an independent study. Whether the observed effects are dependent or independent of vitamin D activation remains to be determined.     

Functional expression of the plant K channel KAT1 in insect cells

Following the biophysical analysis of plant K⁺ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K⁺ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K⁺-selective (impermeable to Na⁺ and even NH₄⁺) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca²⁺ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.     

Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ～1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-β-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.     

Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

Selection of biologically active peptides by phage display of random peptide libraries

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein—protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.     

Das elektronenmikroskopische Bild Menschlicher Endometrialer Drüsenzellen Während des Menstruellen Zyklus

Zusammenfassung Die elektronenmikroskopisch sichtbaren Veränderungen menschlicher endometrialer Drüsenzellen im Verlauf des menstruellen Zyklus werden beschrieben.In der Proliferationsphase zeichnen sich die Drüsenzellen durch reichliche Ergastoplasmamembranen und Paladegranula aus, besonders in den basalen Zytoplasmaanteilen. Daneben sieht man, fast ausschließlich supranukleär, zahlreiche Sekretgranula von etwa 0,7 Durchmesser, deren Zahl am Ende der Proliferationsphase ein Maximum erreicht. Außerdem findet man noch am basalen Kernpol ein Sekret, das aus einem elektronenoptisch schwach konturierten Material besteht und aus Glykogen sowie Glyk- ound Mucoproteiden aufgebaut ist. Gleichzeitig werden die hier liegenden Paladegranula und Ergastoplasmamembranen aufgelöst. Die hier liegenden Mitochondrien vergrößern sich auf ein Mehrfaches, die Zahl ihrer Cristae nimmt zu. Sobald die Sekretproduktion abgeschlossen ist, verkleinern sie sich wieder.Zur Zeit der mittleren Sekretionsphase ist dieses Sekret in das apikale Zytoplasma gewandert. Dabei verschwinden die in den vorangehenden Subphasen reichlich vorhandenen Mikrovilli weitgehend. Gegen Ende des menstruellen Zyklus erscheinen die Zellen durch Abstoßung der apikalen Zytoplasmateile im ganzen niedriger. Kurz vor der Desquamation lösen sie sich dann voneinander, wobei sich der Interzellularraum auf ein Mehrfaches verbreitert. Gleichzeitig treten im Zytoplasma Degenerationszeichen wie vakuoläre Umwandlungen von Mitochondrien, Ergastoplasmaräume und Golgizone auf. Außerdem verlieren die Zellorganellen ihre scharfen Konturen, und die bis dahin runden oder ovalen Zellkerne zeigen eine unregelmäßige, teilweise sogar gelappte Begrenzung.Die seitlichen Zellgrenzen verlaufen in den dem Drüsenlumen nahen Abschnitten gerade oder leicht gewunden und besitzen zahlreiche Desmosomen. Weiter basal hingegen weisen sie starke Verzahnungen mit den Naehbarzellen auf, wobei die Desmosomen nur noch sehr selten zu finden sind. Nach Abstoßung der Zellspitzen in der späten Sekretionsphase reicht die Verzahnungszone bis an das Drüsenlumen heran.Die Basalmembran der Drüsen ist zu Beginn des Zyklus relativ schmal (etwa 300 Å). Sie wächst dann in den späteren Subphasen weiter an und erreicht am Ende des Zyklus eine Dicke von etwa 800 Å.Neben den Drüsenzellen begegnet man hin und wieder in allen Subphasen cilientragenden Zellen (Flimmerzellen), die relativ arm an Zytoplasmaorganellen sind. Die Cilien besitzen den typischen Aufbau mit 9 auf einem Kreisbogen liegenden und einem zentralen Filament, die aus je 2 Subfilamenten bestehen.Außerdem sieht man mitunter zwischen den Drüsenzellen einen weiteren Zelltyp, der reich an Paladegranula und Ergastoplasmastrukturen ist. Art und Funktion dieser Zellen, bei denen es sich nicht um Wanderzellen wie Plasmazellen, Lympho- oder Leukozyten handelt, ist noch unklar.Herrn Prof. Dr. med. H. Siebke und Herrn Oberarzt Doz. Dr. Puck, Universitäts-Frauenklinik Bonn, danke ich für Überlassung des Untersuchungsgutes, Herrn Prof. Dr. med. Piekarski, Hygiene-Institut der Universität Bonn, für die Benutzung des Siemens-Elmiskops.