Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland

Ghrelin (G-HH) synthesized in several tissues including salivary and stomach glands stimulates appetite in humans by modulating neuropeptide Y neurons in the hypothalamic arcuate nucleus. Loss of appetite is one of the most important symptoms of stomach cancer. We conducted a study using immunohistochemistry to determine whether salivary glands and stomach cancer tissues produce ghrelin. We determined that negative ghrelin immunohistochemistry discriminates tumors from normal tissues and may therefore further our understanding of the clinically important problem of reduced food intake and anorexia in cancer patients. Radioimmunoassay analyses confirmed that cancer cells do not produce a G-HH peptide, whereas normal cells yield this peptide.     

Synthesis of a heterotrifunctional linker for the site-specific preparation of antibody-drug conjugates with two distinct warheads

Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.     

A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein

The trypanosome variant surface glycoprotein (VSG), like many other eukaryotic cell surface proteins, is anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) moiety. This glycolipid is assembled first as a precursor (glycolipid A) that is then covalently attached to the newly synthesized polypeptide. We have developed a trypanosome cell-free system capable of performing all of the steps in the biosynthesis of the glycan portion of glycolipid A. Using [3H]sugar nucleotides as substrates, several biosynthetic intermediates have been identified. From structural analyses of these intermediates, we propose a pathway for GPI biosynthesis. Based on comparisons between the VSG GPI anchor and similar structures in other cells, we believe that this same pathway will apply to the GPI anchors, and the related insulin-mediator compound, of higher eukaryotes.     

Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1 E4 protein is lost during malignant progression, but in premalignant lesions, E1 E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 E4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1 E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1 E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1 E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 E4-expressing cells. We hypothesize that E1 E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 E4 expression may contribute to malignant progression.     

Some genetic aspects of the symbiotic relationship between white clover (Trifolium repens) and Rhizobium Trifolii

Summary The results of experiments with white clover (Trifolium repens) in which time of nodulation and seedling plant weight or vigour were measured are reported. Experiments 1 and 2 were conducted in artificial growth medium in test tubes with controlled inoculation and experiment 3 in soil without controlled inoculation. Experiment 1 which was preliminary in nature showed the extent of the variation for time of nodulation after inoculation with Rhizobium trifolii. It was evident also that plant vigour and the number of days to nodulation were negatively correlated. Experiment 2 forms the major part of the results and is concerned with the analyses and interpretation of the diallel cross progeny of twelve plants selected from experiment 1. The results indicated a rather complex genetic picture for the two characters measured, namely days to nodulation and seedling plant weight at 80 days. Reciprocal (both general and specific) as well as additive (g.c.a.) and non-additive (s.c.a.) effects were present. Experiment 3, in which seed from 22 families of the diallel cross was sown in soil without controlled inoculation, indicated that the results obtained under the laboratory conditions of experiment 2 and those obtained in soil conditions were not correlated. The implications of these results in relation to selection of improved varieties of the host species are discussed.

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Zusammenfassung In drei Versuchen wurde beim Weißklee (Trifolium repens) die Zeit bis zur Knöllchenbildung sowie das Gewicht der Pflanze bzw. deren Entwicklungsgeschwindigkeit ermittelt. Bei Versuch 1 und 2 erfolgte die Kultur in Reagenzgläsern auf künstlichem Nährboden nach kontrollierter Impfung und bei Versuch 3 im Boden ohne Impfung. Zunächst wurde im Versuch 1 die Variabilität der Zeitdauer bis zur Knöllchenbildung nach Impfung mit Rhizobium trifolii untersucht, und es zeigte sich eine auffällige Beziehung zwischen der Entwicklungsgeschwindigkeit der Knollen und der Pflanze. Den wesentlichsten Teil der Ergebnisse lieferte Versuch 2, in dem die Nachkommenschaften dialleler Kreuzungen von 12 in Versuch 1 selektierten Pflanzen hinsichtlich der Kriterien Anzahl Tage bis zur Knöllchenbildung und Pflanzengewicht nach 80 Tagen geprüft wurden. Die Resultate ergaben ein recht kompliziertes Bild von der genetischen Veranlagung der beiden untersuchten Merkmale. Es wurden reziproke Unterschiede (sowohl allgemein wie spezifisch) sowie additive und nicht-additive Wirkungen festgestellt. Schließlich wurden im Versuch 3 die Nachkommenschaften aus 22 diallelen Kreuzungen in Erde ohne Impfung geprüft und die Ergebnisse mit denen aus Versuch 2 verglichen. Es wurde festgestellt, daß sie nicht übereinstimmten.

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Zum Schluß wird die Bedeutung dieser Ergebnisse im Hinblick auf die Selektion von verbesserten Formen von Wirtspflanzen diskutiert.