Generation and analysis of p23- and calnexin-deficient mice

This thesis provides in vivo information about two proteins - p23 and calnexin - which play major roles in the early secretory pathway of mammalian cells. The aim was to gain further understanding about the function of these proteins by generating mice deficient in the expression of p23 and calnexin. p23 is a member of the integral p24 protein family. These proteins are highly abundant in the endoplasmic reticulum and Golgi apparatus and are thought to play a role in protein transport and vesicle formation. Disruption of both p23 alleles results in early embryonic lethality. Inactivation of one allele leads not only to reduced levels of p23 itself but also of other family members. The reduction in steady state protein levels also leads to an altered subcellular distribution of p23 as well as p26 (another family member) in p23 heterozygous cells. In addition, structural changes in the Golgi apparatus, in particular dilated saccules, were observed. These changes in p23 heterozygous mice have functional consequences, resulting in specific defects in the secretion of some plasma proteins. Calnexin is a molecular chaperone molecule which is involved in the correct folding of newly synthesised polypeptide chains in the ER. 40% of the calnexin homozygous deficient mice died shortly after birth, the remaining 60% developed a severe neuronal phenotype and had to be sacrificed within 2 months. They were about one third smaller than their littermates and displayed motor disorders which included shaking of the body and a wobbly unsteady gait. Histological examination of brain, cerebellum, spinal cord, neuromuscular junctions and muscle did not reveal any abnormalities in the calnexin-deficient mice. Although these mice showed clear neurodegenerative symptoms, the molecular basis for the phenotype has not yet been characterised

Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice

AbstractDNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1,2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4,5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3,6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8–10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11–14], DNA ligase IV has an essential function in early mammalian development

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Early Postnatal Death and Motor Disorders in Mice Congenitally Deficient in Calnexin Expression

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific