Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope

The nuclear envelope consists of a doublemembraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules

Identifying Novel Leads Using Combinatorial Libraries: Issues and Successes

Chemically generated libraries of small, non-oligomeric compounds are being widely embraced by researchers in both industry and academia. There has been a steady development of new chemistries and equipment applied to library generation so it is now possible to synthesize almost any desired class of compound. However, there are still important issues to consider that range from what specific types of compounds should be made to concerns such as sample resynthesis, structural confirmation of the hit identified, and how to best integrate this technology into a pharmaceutical drug discovery operation. This paper illustrates our approach to new lead discovery (individual, diverse, drug-like molecules of known structural identity using a simple, spatially addressable parallel synthesis approach to prepare Multiple Diverse as well as Universal Libraries) and describes some representative examples of chemistries we had developed within these approaches (preparation of bis-benzamide phenols, thiophenes, pyrrolidines, and highly substituted biphenyls). Finally, the manuscript concludes by addressing some the present concerns that still must be considered in this field

Vaccinia Virus Gene B7R Encodes an 18-kDa Protein That is Resident in the Endoplasmic Reticulum and Affects Virus Virulence

AbstractThis paper presents a characterisation of vaccinia virus (VV) gene B7R that was predicted to encode a polypeptide of 182 amino acids with an N-terminal signal peptide. In vitro transcription and translation analysis showed the B7R gene product was a 21-kDa protein that, in the presence of microsomes, was processed into an 18-kDa mature form. The 18-kDa form associated with the microsomal membranes and was within the lumen of the vesicle where it was inaccessible to exogenous protease or an antibody raised against the B7R C terminus. Within VV-infected cells, the 18-kDa form of B7R was detected late during infection in the endoplasmic reticulum where it colocalised with protein disulphide isomerase. The B7R protein was detected neither in the culture supernatant nor associated with virus particles. A virus deletion mutant lacking the B7R gene and a revertant virus were constructed. Compared to wild-type and revertant viruses, the deletion mutant replicated normally in cell culture and had unaltered virulence in a murine intranasal model of infection. However, the deletion mutant was attenuated in a murine intradermal model where it induced a smaller lesion than the control viruses

Don’t Worry, Be Happy: The Gettability of Ultimate Meaning

Rivka Weinberg advances an error theory of ultimate meaning with three parts: (1) a conceptual analysis, (2) the claim that the extension of the concept is empty, and (3) a proposed fitting response, namely being very, very sad. Weinberg’s conceptual analysis of ultimate meaning involves two features that jointly make it metaphysically impossible, namely (i) the separateness of activities and valued ends, and (ii) the bounded nature of human lives. Both are open to serious challenges. We offer an internalist alternative to (i) and a relational alternative to (ii). We then draw out implications for (2) and conclude with reasons to be cheerful about the prospects of a meaningful life

Don’t Worry, Be Happy: The Gettability of Ultimate Meaning

Rivka Weinberg advances an error theory of ultimate meaning with three parts: (1) a conceptual analysis, (2) the claim that the extension of the concept is empty, and (3) a proposed fitting response, namely being very, very sad. Weinberg’s conceptual analysis of ultimate meaning involves two features that jointly make it metaphysically impossible, namely (i) the separateness of activities and valued ends, and (ii) the bounded nature of human lives. Both are open to serious challenges. We offer an internalist alternative to (i) and a relational alternative to (ii). We then draw out implications for (2) and conclude with reasons to be cheerful about the prospects of a meaningful life

A New Inhibitor of Apoptosis from Vaccinia Virus and Eukaryotes

A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses

A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation

The mammalian nuclear lamina protein lamin B1 is posttranslationally modified by farnesylation, endoproteolysis, and carboxymethylation at a carboxyl-terminal CAAX motif. In this work, we demonstrate that the CAAX endoprotease Rce1 is required for lamin B1 endoproteolysis, demonstrate an independent pool of proteolyzed but nonmethylated lamin B1, as well as fully processed lamin B1, in interphase nuclei, and show a role for methylation in the organization of lamin B1 into domains of the nuclear lamina. Deficiency in the endoproteolysis or methylation of lamin B1 results in loss of integrity and deformity of the nuclear lamina. These data show that the organization of the nuclear envelope and lamina is dependent on a mechanism involving the methylation of lamin B1, and they identify a potential mechanism of laminopathy involving a B-type lamin

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype