Human Myeloma Marrow Cells in Immunologically Deficient Mice

Intact human bone marrow cells from 7 patients with myelomatosis were inoculated intravenously into adolescent CBA mice rendered immunologically deficient by thymectomy followed by total body irradiation (600 rad). Each inoculum of human myeloma marrow cells and subsequent passages of intact mouse marrow and spleen cells resulted in the presence of morphological changes in the marrow, spleen and peripheral blood of a proportion of these mice which were closely similar to those seen in the human donor. A substantial amount of human immunoglobulin (IgG and IgA) was detected in the sera of some of the mice showing morphological changes. Mice prepared identically but remaining uninoculated or receiving intact human bone marrow cells from 3 patients with no evidence of haematological malignancy showed none of these changes when examined after similar intervals

The molecular mechanism of DNA damage recognition by MutS homologs and its consequences for cell death response

We determined the molecular mechanism of cell death response by MutS homologs in distinction to the repair event. Key protein–DNA contacts differ in the interaction of MutS homologs with cisplatinated versus mismatched DNA. Mutational analyses of protein–DNA contacts, which were predicted by molecular dynamics (MD) simulations, were performed. Mutations in suggested interaction sites can affect repair and cell death response independently, and to different extents. A glutamate residue is identified as the key contact with cisplatin-DNA. Mutation of the residue increases cisplatin resistance due to increased non-specific DNA binding. In contrast, the conserved phenylalanine that is instrumental and indispensable for mismatch recognition during repair is not required for cisplatin cytotoxicity. These differences in protein–DNA interactions are translated into localized conformational changes that affect nucleotide requirements and inter-subunit interactions. Specifically, the ability for ATP binding/hydrolysis has little consequence for the MMR-dependent damage response. As a consequence, intersubunit contacts are altered that most likely affect the interaction with downstream proteins. We here describe the interaction of MutS homologs with DNA damage, as it differs from the interaction with a mismatch, and its structural translation into all other functional regions of the protein as a mechanism to initiate cell death response and concomitantly inhibit repair

Bigger is not always better : viability selection on body mass varies across life stages in a hibernating mammal

ACKNOWLEDGEMENTS: We would like to express our thanks to all the hard-working marmoteers, across the course of the study, that helped to collect the annual field data. In addition, we would like to specifically thank Kenneth B. Armitage for starting the project and access to the long-term body mass data. This work 431 was supported by an EASTBIO PhD studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) and the University of Aberdeen, which was awarded to A.H.M.J. D.T.B was supported by the National Geographic Society, UCLA (Faculty Senate and the Division of Life Sciences), a Rocky Mountain Biological Laboratory research fellowship, and NSF-IDBR-0754247, DEB435 1119660 and 1557130 (to DTB); and NSF-DBI 0242960, 0731346, and 1262713 (to the RMBL).Peer reviewedPublisher PD

Parameters of Reserpine Analogs That Induce MSH2/MSH6-Dependent Cytotoxic Response

Mismatch repair proteins modulate the cytotoxicity of several chemotherapeutic agents. We have recently proposed a “death conformation” of the MutS homologous proteins that is distinguishable from their “repair conformation.” This conformation can be induced by a small molecule, reserpine, leading to DNA-independent cell death. We investigated the parameters for a small reserpine-like molecule that are required to interact with MSH2/MSH6 to induce MSH2/MSH6-dependent cytotoxic response. A multidisciplinary approach involving structural modeling, chemical synthesis, and cell biology analyzed reserpine analogs and modifications. We demonstrate that the parameters controlling the induction of MSH2/MSH6-dependent cytotoxicity for reserpine-analogous molecules reside in the specific requirements for methoxy groups, the size of the molecule, and the orientation of molecules within the protein-binding pocket. Reserpine analog rescinnamine showed improved MSH2-dependent cytotoxicity. These results have important implications for the identification of compounds that require functional MMR proteins to exhibit their full cytotoxicity, which will avoid resistance in MMR-deficient cells

Modelling autosomal dominant optic atrophy associated with OPA1 variants in iPSC-derived retinal ganglion cells

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the preferential loss of retinal ganglion cells (RGCs), resulting in optic nerve degeneration and progressive bilateral central vision loss. Over 60% of genetically confirmed DOA patients carry variants in the nuclear OPA1 gene, which encodes for a ubiquitously expressed, mitochondrial GTPase protein. OPA1 has diverse functions within the mitochondrial network, facilitating inner membrane fusion and cristae modelling, regulating mitochondrial DNA maintenance and coordinating mitochondrial bioenergetics. There are currently no licensed disease-modifying therapies for DOA and the disease mechanisms driving RGC degeneration are poorly understood. Here, we describe the generation of isogenic, heterozygous OPA1 null iPSC (OPA1+/-) through CRISPR/Cas9 gene editing of a control cell line, in conjunction with the generation of DOA patient-derived iPSC carrying OPA1 variants, namely, the c.2708_2711delTTAG variant (DOA iPSC), and previously reported missense variant iPSC line (c.1334G>A, DOA+ iPSC) and CRISPR/Cas9 corrected controls. A two-dimensional (2D) differentiation protocol was used to study the effect of OPA1 variants on iPSC-RGC differentiation and mitochondrial function. OPA1+/-, DOA and DOA+ iPSC showed no differentiation deficit compared to control iPSC lines, exhibiting comparable expression of all relevant markers at each stage of differentiation. OPA1+/- and OPA1 variant iPSC-RGCs exhibited impaired mitochondrial homeostasis, with reduced bioenergetic output and compromised mitochondrial DNA maintenance. These data highlight mitochondrial deficits associated with OPA1 dysfunction in human iPSC-RGCs, and establish a platform to study disease mechanisms that contribute to RGC loss in DOA, as well as potential therapeutic interventions

EMCS Retrofit Analysis - Interim Report

This report presents the interim results of analyses carried out in the Phillip Burton Federal Building in San Francisco from 1996 to 1998. The building is the site of a major demonstration of the BACnet communication protocol. The energy management and control systems (EMCS) in the building were retrofitted with BACnet compatible controllers in order to integrate certain existing systems on one common network. In this respect, the project has been a success. Interoperability of control equipment from different manufacturers has been demonstrated in a real world environment. Besides demonstrating interoperability, the retrofits carried out in the building were also intended to enhance control strategies and capabilities, and to produce energy savings. This report presents analyses of the energy usage of HVAC systems in the building, control performance, and the reaction of the building operators. The report does not present an evaluation of the performance capabilities of the BACnet protocol. A monitoring system was installed in the building that parallels many of the EMCS sensors and data were archived over a three-year period. The authors defined pre-retrofit and post-retrofit periods and analyzed the corresponding data to establish the changes in building performance resulting from the retrofit activities. The authors also used whole-building energy simulation (DOE-2) as a tool for evaluating the effect of the retrofit changes. The results of the simulation were compared with the monitored data. Changes in operator behavior were assessed qualitatively with questionnaires. The report summarizes the findings of the analyses and makes several recommendations as to how to achieve better performance. They maintain that the full potential of the EMCS and associated systems is not being realized. The reasons for this are discussed along with possible ways of addressing this problem. They also describe a number of new technologies that could benefit systems of the type found in the Philip Burton Federal Building