Evolution of bacterial pathogenesis

The evolution of bacteria is associated with continuous generation of novel genetic variants. The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer. A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease. Additionally, many bacteria express resistance traits against antibiotics. Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation. They are often encoded on unstable DNA regions. Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes. This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties. In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens.     

Signalling via caveolin: involvement in the cross-talk between phosphoinositolglycans and insulin

In recent years, a number of cross-talk systems have been identified which feed into the insulin signalling cascade at the level of insulin receptor substrate (IRS) tyrosine phosphorylation, e.g., receptor and non-receptor tyrosine kinases and G-protein-coupled receptors. At the molecular level, a number of negative modulator and feedback systems somehow interacting with the beta-subunit (catecholamine-, phorbolester-, or tumor necrosis factor-alpha-induced serine/threonine phosphorylation, carboxy-terminal trimming by a thiol-dependent protease, association of inhibitory/regulatory proteins such as RAD, PC1, PED, alpha2-HS-glycoprotein) have been identified as candidate mechanisms for the impairment of insulin receptor function by elevations in the activity and/or amount of the corresponding modification enzymes/inhibitors. Both decreased responsiveness and sensitivity of the insulin receptor beta-subunit for insulin-induced tyrosine autophosphorylation have been demonstrated in several cellular and animal models of metabolic insulin resistance as well as in the adipose tissue and skeletal muscle of diabetic patients and obese Pima Indians compared to non-obese subjects. Therefore, induction of the insulin signalling cascade by bypassing the defective insulin receptor kinase may be useful for the therapy of non-insulin dependent diabetes mellitus. During the past two decades, phosphoinositolglycans (PIGs) of various origin have been demonstrated to exert potent insulin-mimetic metabolic effects upon incubation with cultured or isolated muscle and adipose cells. However, it remained to be elucidated whether these compounds actually manage to trigger insulin signalling and if so at which level of hierarchy within the signalling cascade the site of interference is located. Recent studies using isolated rat adipocytes and chemically synthesized PIG compounds point to IRS1/3 tyrosine phosphorylation by p59Lyn kinase as the site of cross-talk, the negative regulation of which by interaction with caveolin is apparently abrogated by PIG. This putative mechanism is thus compatible with the recently formulated caveolin signalling hypothesis, the supporting data for which are reviewed here. Though we have not obtained experimental evidence for the involvement of PIG in physiological insulin action, the potential cross-talk between insulin and PIG signalling, including the caveolae/detergent-insoluble glycolipid-enriched rafts as the compartments where the corresponding signalling components are concentrated, thus represent novel targets for signal transduction therapy.     

Proteasomes in apoptosis: villains or guardians?

The proteasome (multicatalytic proteinase complex, prosome) is a major cytoplasmic proteolytic enzyme, responsible for degradation of the vast majority of intracellular proteins. Proteins degraded by the proteasome are usually tagged with multiple ubiquitin moieties, conjugated to the substrates by a complicated cascade of enzymes. Over the last years, evidence has accumulated that changes in the expression and activity of the different components of the ubiquitin-proteasome system occur during apoptosis. Proteasome inhibitors have been used to induce apoptosis in various cell types, whereas in others, these compounds were able to prevent apoptosis induced by different stimuli. The proteasome mediated step(s) in apoptosis is located upstream of mitochondrial changes and caspase activation, and can involve in different systems Bcl-2, Jun N-terminal kinase, heat shock proteins, Myc, p53, polyamines and other factors.     

High-resolution mapping of quantitative trait loci in outbred mice

Screening the whole genome of a cross between two inbred animal strains has proved to be a powerful method for detecting genetic loci underlying quantitative behavioural traits, but the level of resolution offered by quantitative trait loci (QTL) mapping is still too coarse to permit molecular cloning of the genetic determinants. To achieve high-resolution mapping, we used an outbred stock of mice for which the entire genealogy is known. The heterogeneous stock (HS) was established 30 years ago from an eight-way cross of C57BL/6, BALB/c, RIII, AKR, DBA/2, I, A/J and C3H inbred mouse strains. At the time of the experiment reported here, the HS mice were at generation 58, theoretically offering at least a 30-fold increase in resolution for QTL mapping compared with a backcross or an F2 intercross. Using the HS mice we have mapped a QTL influencing a psychological trait in mice to a 0.8-cM interval on chromosome 1. This method allows simultaneous fine mapping of multiple QTLs, as shown by our report of a second QTL on chromosome 12. The high resolution possible with this approach makes QTLs accessible to positional cloning.     

Notch signalling pathway mediates hair cell development in mammalian cochlea

The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.     

Chemotherapy and immunotherapy of malignant glioma: molecular mechanisms and clinical perspectives

Despite the considerable progress in modern tumor therapy, the prognosis for patients with glioblastoma, the most frequent malignant brain tumor, has not been substantially improved. Although cytoreductive surgery and radiotherapy are the mainstays of treatment for malignant glioma at present, novel cytotoxic drugs and immunotherapeutic approaches hold great promise as effective weapons against these malignancies. Thus, great efforts are being made to enhance antitumoral efficacy by combining various cytotoxic agents, by novel routes of drug administration, or by combining anticancer drugs and immune modulators. Immunotherapeutic approaches include cytotoxic cytokines, targeted antibodies, and vaccination strategies. However, the success of most of these experimental therapies is prevented by the marked molecular resistance of glioma cells to diverse cytotoxic agents or by glioma-associated immunosuppression. One promising experimental strategy to target glioma is the employment of death ligands such as CD95 (Fas/Apo1) ligand or Apo2 ligand (TRAIL). Specific proapoptotic approaches may overcome many of the obvious obstacles to a satisfactory management of malignant brain tumors. Received 8 March 1999; received after revision 27 May 1999; accepted 14 June 1999     

Genomic instability in Gadd45a-deficient mice.

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.     

Engineering a mouse balancer chromosome.

Balancer chromosomes are genetic reagents that are used in Drosophila melanogaster for stock maintenance and mutagenesis screens. Despite their utility, balancer chromosomes are rarely used in mice because they are difficult to generate using conventional methods. Here we describe the engineering of a mouse balancer chromosome with the Cre-loxP recombination system. The chromosome features a 24-centiMorgan (cM) inversion between Trp53 (also known as p53) and Wnt3 on mouse chromosome 11 that is recessive lethal and dominantly marked with a K14-Agouti transgene. When allelic to a wild-type chromosome, the inversion suppresses crossing over in the inversion interval, accompanied by elevated recombination in the flanking regions. The inversion functions as a balancer chromosome because it can be used to maintain a lethal mutation in the inversion interval as a self-sustaining trans-heterozygous stock. This strategy can be used to generate similar genetic reagents throughout the mouse genome. Engineering of visibly marked inversions and deficiencies is an important step toward functional analyses of the mouse genome and will facilitate large-scale mutagenesis programs.     

CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome.

Altered growth and function of synoviocytes, the intimal cells which line joint cavities and tendon sheaths, occur in a number of skeletal diseases. Hyperplasia of synoviocytes is found in both rheumatoid arthritis and osteoarthritis, despite differences in the underlying aetiologies of the two disorders. We have studied the autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; MIM 208250) to identify biological pathways that lead to synoviocyte hyperplasia, the principal pathological feature of this syndrome. Using a positional-candidate approach, we identified mutations in a gene (CACP) encoding a secreted proteoglycan as the cause of CACP. The CACP protein, which has previously been identified as both 'megakaryocyte stimulating factor precursor' and 'superficial zone protein', contains domains that have homology to somatomedin B, heparin-binding proteins, mucins and haemopexins. In addition to expression in joint synovium and cartilage, CACP is expressed in non-skeletal tissues including liver and pericardium. The similarity of CACP sequence to that of other protein families and the expression of CACP in non-skeletal tissues suggest it may have diverse biological activities.