Mapping of the leptin binding sites and design of a leptin antagonist

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.     

A new alkali-thermostable azoreductase from Bacillus sp. strain SF

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain, Bacillus sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80 degrees C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO2 substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.     

Characterization of central nervous system structures by magnetic resonance diffusion anisotropy

Diffusion-weighted magnetic resonance imaging (MRI) provides information about tissue water diffusion. Diffusion anisotropy, which can be measured with diffusion tensor MRI, is a quantitative measure of the directional dependence of the diffusion restriction that is introduced by biological structures such as nerve fibers. Diffusion tensor MRI data was obtained in the brain, brain stem, and cervical spinal cord. For each region, scans were performed in four normal volunteers. Fractional anisotropy (FA), an index of diffusion anisotropy, was measured within regions of interest located in the corpus callosum, capsula interna, thalamus, caudate nucleus, putamen, brain cortex, pyramidal tract of the medulla, accessory olivary nucleus, dorsal olivary nucleus, inferior olivary nucleus, spinal white and gray matter. The highest FA value was measured in the corpus callosum (81 +/- 3%). The values of the other areas decreased in the following order: pyramidal tract in the medulla (72 +/- 1%), spinal white matter (65 +/- 4%), capsula interna (62 +/- 3%), accessory olivary nucleus (36 +/- 2%), spinal gray matter (35 +/- 5%), dorsal olivary nucleus in the medulla (29 +/- 2%), thalamus (28 +/- 2%), inferior olivary nucleus (15 +/- 2%), putamen (13 +/- 2%), caudate nucleus (13 +/- 2%), and brain cortex (9 +/- 1%). Our results indicate that the underlying fiber architecture, fiber density, and uniformity of nerve fiber direction affect anisotropy values of the various structures. Characterization of various central nervous system structures with diffusion anisotropy is possible and may be useful to monitor degenerative diseases in the central nervous system.     

Glial activation and pathological pain

Pain is a sensation we have all experienced. For most of us, the pain has been temporary. However, for patients with pathological pain, the pain experience is unending, with little hope for therapeutic relief. Pathological pain is characterized by an amplified response to normally innocuous stimuli, and an amplified response to acute pain. Pathological pain has long been described as the result of dysfunctional neuronal activity. While neuronal functioning is indeed altered, there is significant evidence showing that exaggerated pain is regulated by the activation of astrocytes and microglia. In exaggerated pain, astrocytes, and microglia are activated by neuronal signals including substance P, glutamate, and fractalkine. Activation of glia by these substances leads to the release of mediators that then act on other glia and neurons. These include a family of proteins called "proinflammatory cytokines" released from microglia and astrocytes. These cytokines have been shown to be critical mediators of exaggerated pain. Some patients with pathological pain also report "extra-territorial" and/or "mirror" image pain. That is, exaggerated pain is experienced not only in the area of trauma. In extra-territorial pain, pain is also perceived as arising from neighboring healthy tissues outside of the site of trauma. In the rare cases of mirror-image pain, such pain is perceived as arising from the healthy, corresponding body part on the opposite side of the body. New data suggest that activation of astrocyte communication via gap junctions may mediate such spread of pain. While traditional therapies for pathological pain have focused on neuronal targets, the following review describes glia as newly recognized mediators of exaggerated pain, and as new therapeutic targets. Moreover, the glial-neuronal interactions discussed here are likely not exclusive to pain, but rather are likely to play significant roles in other behavioral phenomena.     

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.     

Androgen and Corticoid Levels in Blood and Body Extracts of High- and Low-ranking Swordtail Males (Xiphophorus helleri) before and after Social Isolation

The two highest- and the two lowest-ranking swordtail males were determined by the criterion of number of attacks directed at and received from every other male in their breeding tanks. The events were recorded until 50 bites were given (high-ranking males) or received (low-ranking males). 40 high- and 40 low-ranking males were bled either after being socially isolates for 4 weeks or after reaching the target number. Androgen and corticoid concentrations both in their blood and their body extracts were measured by means of radioimmunoassay. Although the agonistic behaviour patterns and social experience of high- and low-ranking males were totally different, the baseline hormonal states of the animals were indistinguishable both in body extracts and blood. Isolation led to decreased blood androgen and corticoid levels in high- and low-ranking males. In the body extracts, only the corticoids were reduced. A significant correlation between blood and tissue concentrations was found only in the case of corticoids. I would suggest that the contradictory findings in the literature dealing with the relationship between hormones and dominance result from differences in experimental procedure.     

A cradle for new proteins: trigger factor at the ribosome

Newly synthesized proteins leave the ribosome through a narrow tunnel in the large subunit. During ongoing synthesis, nascent protein chains are particularly sensitive to aggregation and degradation because they emerge from the ribosome in an unfolded state. In bacteria, the first protein to interact with nascent chains and facilitate their folding is the ribosome-associated chaperone trigger factor. Recently, crystal structures of trigger factor and of its ribosome-binding domain in complex with the large ribosomal subunit revealed that the chaperone adopts an extended 'dragon-shaped' fold with a large hydrophobic cradle, which arches over the exit of the ribosomal tunnel and shields newly synthesized proteins. These structural results, together with recent biochemical data on trigger factor and its interplay with other chaperones and factors that interact with the nascent chain, provide a comprehensive view of the role of trigger factor during co-translational protein folding.     

Gigaxonin interacts with tubulin folding cofactor B and controls its degradation through the ubiquitin-proteasome pathway

Gigaxonin is mutated in human giant axonal neuropathy (GAN), an autosomal recessive neurodegenerative disorder. The presence of generalized cytoskeletal abnormalities , including few microtubules and accumulated intermediate filaments (IFs), in GAN suggests an essential role of gigaxonin in cytoskeletal organization and dynamics. However, the molecular mechanisms underlying the cytoskeletal pathology remain to be elucidated. Over the years, the ubiquitin-proteasome system (UPS) of intracellular protein degradation has been implicated in the control of many fundamental cellular processes. Defects in this system seem to be directly linked to the development of human diseases, including cancers and neurodegenerative diseases . Here, we show that gigaxonin controls protein degradation of tubulin folding cofactor B (TBCB) , a function disrupted by GAN-associated mutations. The substantial TBCB protein accumulation caused by impaired UPS may be a causative factor of cytoskeletal pathology in GAN. Our study provides important insight into pathogenesis of neurodegenerative diseases associated with cytoskeletal abnormalities.     

Juvenile immune system activation induces a costly upregulation of adult immunity in field crickets Gryllus campestris

Inducible immune defence may allow organisms a state-dependent upregulation of costly immunity in order to minimize the risk of anticipated future parasitism. The basic costs of elevated immune activity might involve a reduction in other fitness-related traits as well as an increased risk of immunopathology. In male field crickets Gryllus campestris we experimentally investigated the condition-dependent effects of immune system activation in nymphs on immunity and physiological condition during adulthood. Following a nymphal injection of bacterial lipopolysaccharides, adult males showed significantly elevated levels of two major immune parameters, i.e. haemolymph antibacterial activity and the concentration of prophenoloxidase (proPO). By contrast, the active enzyme, phenoloxidase (PO), did not increase, suggesting a strategic long-term upregulation of the inactive proenzyme proPO only. This may help avoid the cytotoxic effects associated with high standing levels of the active enzyme. The nymphal immune insult further caused a reduction in adult haemolymph protein load, suggesting a long-term decline in overall metabolic condition. Nymphal food availability positively affected adult lysozyme activity, while PO and proPO concentrations were not affected. Our data thus suggest the long-term upregulation of immunity in response to antigenic cues as an adaptive, yet costly, invertebrate strategy to improve resistance to future parasitism.