The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function

Objective. To identify and investigate the kinetic binding properties of interleukin-1 receptors (IL-1R), and examine the abilities of the 2 IL-1 isoforms to stimulate metalloprotease synthesis, in normal and osteoarthritic (OA) chondrocytes. Methods. Receptor affinity and density were determined using radioligand binding experiments and flow cytometry. Immunocytochemical analysis and affinity cross-linking studies were performed for characterization of IL-1R. Results. While no difference in receptor affinity between normal and OA chondrocytes was noted in binding studies (k_d ˜30 pM), a 2-fold increase in receptor density was found in OA chondrocytes as compared with normal chondrocytes (mean 4,069 sites/cell versus 2,315 sites/cell). Flow cytometry experiments also showed a significant increase in receptor density in OA cells, as well as an enhancement in the percentage of positive cells in diseased cartilage compared with normal. Binding data for both IL-1 isoforms revealed a single class of binding sites and receptor specificity. Factors such as IL-2, interferon-sγ, tumor necrosis factor α, and bovine insulin did not compete with IL-1β. By covalent ligand cross-linking and electrophoretic analysis, only type I IL-1R, a protein of 80 kd, was detected on chondrocytes. By immunocytochemical analysis, IL-1R was identified at the cell membrane level, in both normal and OA chondrocytes. The presence of nuclear staining was also observed, but only in OA chondrocytes. Recombinant human IL-1 (α and β) induced the secretion of stromelysin and collagenase in a dose-dependent manner. The IL-1 concentration required for half-maximal metalloprotease stimulation was 3–4 times lower in OA chondrocytes than in normal cells. Conclusion. These results indicate that OA chondrocytes have a higher sensitivity to the stimulation of metalloprotease synthesis by IL-1 than do normal cells. This could be related to the increased levels of IL-1R expressed in the OA cells. The implications of these findings with regard to the possible roles of IL-1 and IL-1R in the pathogenesis of OA are discussed.     

Normal CAG and CCG repeats in the Huntington's disease genes of Parkinson's disease patients

The clinical features of Parkinson's disease, particularly rigidity and bradykinesia and occasionally tremor, are seen in juvenile-onset Huntington's disease. Therefore, the CAG and CCG repeats in the Huntington's disease gene were investigated in 45 Parkinson's disease patients and compared to 40 control individuals. All of the Parkinson's disease chromosomes fell within the normal size ranges. In addition, the distributions of the two repeats in the Parkinson's disease patients did not differ significantly from those of the control population. Therefore, abnormalities of these trinucleotide repeats in the Huntington's disease gene are not likely to contribute to the pathogenesis of Parkinson's disease. © 1995 Wiley-Liss, Inc.     

Pancreatic cancers require autophagy for tumor growth

Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.     

Modulation of cortical excitability and interhemispheric inhibition prior to rhythmic unimanual contractions

The objective of this study was to investigate premotor modulation of motor cortical excitability between rhythmic unimanual finger contractions. Applying TMS at rest prior to an anticipated contraction provides a measure of cortical excitability that reflects premotor modulatory drive and is uncontaminated by the alterations in spinal and cortical excitability that occur during muscle activation. We hypothesized that premotor structures contribute to unimanual movement through the modulation of intracortical and interhemispheric inhibitory circuits within the primary motor cortex and that this premotor modulation would be evident at rest between contractions. Thus, we used transcranial magnetic stimulation (TMS) to assess short interval intracortical inhibition (SICI) and interhemispheric inhibition (IHI) in a 500-ms epoch prior to a planned contraction of the right FDI in 10 participants (21.4±1.9 years). These measures of inhibition were made in three different states: (1) at complete rest (with no plan to contract), (2) at rest between rhythmic contractions, and (3) during low level contractions. Cortical excitability was enhanced prior to a contraction and during a contraction compared to at rest (F((2,18))=758.3, p<0.001). IHI was also increased prior to a contraction compared to at rest and during a contraction while SICI was only reduced during a contraction (F((2,38))=30.3, p<0.001).We used this pre-contraction protocol to investigate the cortical mechanisms of unimanual control. However, this protocol would be a useful tool to investigate any neuromuscular adaptation that may occur as a result of altered premotor modulation of cortical excitability, such as neuromuscular fatigue, training and movement disorders.     

Chromosomal reinsertion of broken RSS ends during T cell development

The V(D)J recombinase catalyzes DNA transposition and translocation both in vitro and in vivo. Because lymphoid malignancies contain chromosomal translocations involving antigen receptor and protooncogene loci, it is critical to understand the types of "mistakes" made by the recombinase. Using a newly devised assay, we characterized 48 unique TCRbeta recombination signal sequence (RSS) end insertions in murine thymocyte and splenocyte genomic DNA samples. Nearly half of these events targeted "cryptic" RSS-like elements. In no instance did we detect target-site duplications, which is a hallmark of recombinase-mediated transposition in vitro. Rather, these insertions were most likely caused by either V(D)J recombination between a bona fide RSS and a cryptic RSS or the insertion of signal circles into chromosomal loci via a V(D)J recombination-like mechanism. Although wild-type, p53, p53 x scid, H2Ax, and ATM mutant thymocytes all showed similar levels of RSS end insertions, core-RAG2 mutant thymocytes showed a sevenfold greater frequency of such events. Thus, the noncore domain of RAG2 serves to limit the extent to which the integrity of the genome is threatened by mistargeting of V(D)J recombination.     

The revolution of whole genome sequencing to study parasites

Genome sequencing has revolutionized the way in which we approach biological research from fundamental molecular biology to ecology and epidemiology. In the last 10 years the field of genomics has changed enormously as technology has improved and the tools for genomic sequencing have moved out of a few dedicated centers and now can be performed on bench-top instruments. In this review we will cover some of the key discoveries that were catalyzed by some of the first genome projects and discuss how this field is developing, what the new challenges are and how this may impact on research in the near future.