Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury

Members of the interleukin-6 (IL-6) family of cytokines have been implicated as major mediators of the response of the adult nervous system to injury. The basis for an interaction of IL-6 cytokines with adult sensory neurones has been established by analysing the levels and distribution of the two signal-transducing receptor subunits, glycoprotein 130 (gp130) and leukaemia inhibitory factor receptor (LIFR), in the dorsal root ganglion (DRG) of male adult rats before and following nerve injury. All sensory neurones express gp130-immunoreactivity (IR) in the cytoplasm and on the plasma membrane. Levels of gp130 and its intracellular distribution remained unchanged up to 14 days following sciatic nerve axotomy. LIFR-IR was largely absent from the cytoplasm and plasma membrane of sensory neurones, but confined almost exclusively to the nuclear compartment. However, following axotomy, punctate cytoplasmic LIFR-IR was detected which persisted up to 28 days following axotomy. The expression of cytoplasmic LIFR 2 days post-axotomy was proportionally greater in a subset of small diameter sensory neurones which expressed either the sensory neuropeptide CGRP or the cell surface marker isolectin B4. The coexpression of gp130 and LIFR in the same intracellular compartment following axotomy conveys potential responsiveness of injured sensory neurones to members of the IL-6 family of cytokines.     

Lithium inhibits aluminum-induced apoptosis in rabbit hippocampus,by preventing cytochrome c translocation,Bcl-2 decrease,Bax elevation and caspase-3 activation

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis-regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), increases levels of the pro-apoptotic Bax, activates caspase-3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum-induced translocation of cytochrome c, and up-regulates Bcl-2 and Bcl-X(L,) down-regulates Bax, abolishes caspase-3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis-controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis-regulatory proteins present in the subcellular organelles of rabbit brain.     

A phenotype-sensitizing Apoe-deficient genetic background reveals novel atherosclerosis predisposition loci in the mouse

Therapeutic intervention for atherosclerosis has predominantly concentrated on regulating cholesterol levels; however, these therapeutics are not efficacious for all patients, suggesting that other factors are involved. This study was initiated to identify mechanisms that regulate atherosclerosis predisposition in mice other than cholesterol level regulation. To do so we performed quantitative trait locus analysis using two inbred strains that each carry the atherosclerosis phenotype-sensitizing Apoe deficiency and that have been shown to have widely disparate predilection to atherosclerotic lesion formation. One highly significant locus on chromosome 10 (LOD = 7.8) accounted for 19% of the variance in lesion area independent of cholesterol. Two additional suggestive loci were identified on chromosomes 14 (LOD = 3.2) and 19 (LOD = 3.2), each accounting for 7-8% of the lesion variance. In all, five statistically significant and suggestive loci affecting lesion size but not lipoprotein levels were identified. Many of these were recapitulated in an independent confirmatory cross. In summary, two independently performed crosses between C57BL/6 and FVB/N Apoe-deficient mice have revealed several previously unreported atherosclerosis susceptibility loci that are distinct from loci linked to lipoprotein levels.     

Morphology of cell injury: An approach to the EDIT programme by the use of tobacco pollen tubes. Evaluation-guided Development of New In Vitro Test Batteries

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.     

Neural correlates of target choice for pursuit and saccades in the primate superior colliculus

We have examined the role of the superior colliculus (SC) in choosing targets for pursuit and saccades by comparing neuronal activity at sites representing the possible choices. After recording during a two-alternative forced-choice paradigm, we measured the difference in activity of the populations representing the two choices by computing receiver operating characteristic (ROC) curves on a millisecond timescale. A signal indicating the correct choice emerged from noise over time, forming a tradeoff between speed and accuracy. The observed performance corresponded to particular points along the predicted speed-accuracy curves-pursuit emphasizing speed and saccades emphasizing accuracy. These results show that activity from the same set of neurons in the superior colliculus can predict target choices for both pursuit and saccades.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.     

Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by one million-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species.     

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.     

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.