Liver damage from cocaine in mice

Four daily injections of 20 mg per kg cocaine hydrochloride into B6AF₁/J mice produced focal necrosis of liver parenchymal cells in the midzonal region. Massive liver damage and marked elevation of serum glutamic-oxaloacetic transaminase was observed after a single injection of 50 mg per kg cocaine-HC1. This damage led to increased sleep time after pentobarbital, and a decreased rate of pentobarbital metabolism $\text{in vivo}$.     

EFFECTS OF CHRONIC STEROID INGESTION ON GASTRODUODENAL EPITHELIAL RENEWAL IN THE RAT

Steroids may predispose to peptic ulcer formation. One possible mechanism could be via alteration of normal epithelial renewal. to study the effects of steroids on gastroduodenal epithelial renewal, rats received hydrocortisone sodium succinate in the drinking water to deliver a dose of 10 mg/kg per day. Control rats received plain water. After 4 weeks, the rats were injected intraperitoneally with tritiated thymidine, to label proliferating cells, and killed 1 hr later, to determine measurements of epithelial proliferation, or 24 hr later to determine measurements of epithelial migration. Sections of fundus, antrum and post-pyloric duodenum were processed for light microscopy and autoradiography. In fundic and antral mucosa, steroid treatment resulted in a reduction in the number of labelled cells and in the size of the proliferative zone and, in the fundus, the mucosal thickness was reduced. In the duodenum, although the number of labelled cells remained unchanged, steroid treatment did decrease the number of cells in the proliferative zone; further, crypt depth was reduced in steroid-treated rats, but villous height was increased, resulting in an overall increase in mucosal thickness. Epithelial migration was also depressed in fundic and antral mucosa, but appeared to be accelerated in the duodenum of steroid-treated rats. These studies indicate that although, in the duodenum, the effects of steroids on epithelial renewal are complex, in the stomach chronic steroid ingestion depresses epithelial renewal both in fundic and antral mucosa. This inhibition of epithelial renewal in the stomach may contribute to the ulcerogenic action of steroids by either rendering the mucosa susceptible to the effects of other ulcerogens or by retarding the healing of existing mucosal lesions.     

Sub-chronic administration of the 11beta-HSD1 inhibitor, carbenoxolone, improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.     

IL-1F5 mediates anti-inflammatory activity in the brain through induction of IL-4 following interaction with SIGIRR/TIR8

Similarity in structure and sequence homology has led to the identification of new members of the interleukin-1 (IL-1) ligand and receptor superfamilies. IL-1F6, IL-1F8 and IL-1F9 have been shown to signal through IL-1R-related protein 2 and IL-1 receptor accessory protein leading to activation of NFκB, while IL-1F7 and IL-1F10 interact with the IL-18 receptor and the soluble IL-1 receptor type I respectively. In contrast, identification of a biological role for IL-1F5 has remained elusive, with conflicting data relating to its possible ability to antagonize IL-1F9-stimulated activation of NFκB in Jurkat cells transfected with IL-1R-related protein 2. In this study, we set out to investigate a possible role for IL-1F5 in the brain and report that it antagonizes the inflammatory effects of IL-1β and lipopolysaccharide (LPS) in vivo and in vitro including the inhibitory effect on long-term potentiation (LTP) in rat hippocampus. We demonstrate that IL-1F5 induces IL-4 mRNA and protein expression in glia in vitro and enhances hippocampal expression of IL-4 following intracerebroventricular (i.c.v.) injection. The inhibitory effect of IL-1F5 on LPS-induced IL-1β is attenuated in cells from IL-4-defective (IL−4^−/− mice). Our findings suggest that IL-1F5 mediates anti-inflammatory effects through its ability to induce IL-4 production and that this is a consequence of its interaction with the orphan receptor, single Ig IL-1R-related molecule (SIGIRR)/TIR8, as the effects were not observed in SIGIRR^−/− mice. In contrast to its effects in brain tissue, IL-1F5 did not attenuate LPS-induced changes, or up-regulated IL-4 in macrophages or dendritic cells, suggesting that the effect is confined to the brain.     

Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B′/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC₅₀) and IC₉₀ values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.     

Stress-responsive alpha-dioxygenase expression in tomato roots

Alpha-dioxygenase (alpha-DOX) enzymes catalyse the oxygenation of fatty acids to yield a newly identified group of oxylipins that play a role in protecting tissues from oxidative damage and cell death. In tomato (Lycopersicon esculentum Mill.) alpha-DOX was identified as salt-regulated by differential display of mRNA, and is represented by a small gene family comprising at least three members: LEalpha-DOX1, -2, and -3 of which only LEalpha-DOX1 was salt-responsive. The enhancement of LEalpha-DOX1 expression in roots by salt, wounding and challenge with Pythium aphanidermatum (Edson) Fitzp. suggests that alpha-DOX-generated oxylipins may mediate the response of roots to these environmental stresses. In roots, LEalpha-DOX1 was abscisic acid (ABA)-responsive. However, in the ABA-deficient mutant flacca salt-responsive expression was equivalent to that in the wild type. Similarly, in roots exposed to fluridone (FLU) salt up-regulated expression; however, in this case salt-responsive LEalpha-DOX1 expression was greater than that in roots that were not exposed to FLU. A possible explanation for this is provided by the role of ABA in suppressing ethylene accumulation in osmotically stressed roots. The ethylene-generating agent ethephon and precursor 1-aminocyclopropane-1-carboxylic acid markedly elevated LEalpha-DOX1 expression, and this enhanced expression was suppressed by ABA. LEalpha-DOX1 expression in salt-stressed roots was not markedly affected by AVG indicating that ABA may be responsible for enhanced alpha-DOX expression in salt-treated roots.     

Walking on a ‘peg leg’: extensor muscle activities and sensory feedback after distal leg denervation in cockroaches

Previous studies in insects demonstrated that leg coordination changes following complete ablation of distal limb segments. However, normal coordination was restored when small peg leg prostheses were attached to leg stumps to permit substrate contact. We have adapted this paradigm to preserve appropriate leg mass and inertia by severing all nerves and muscle tendons in the femur of the cockroach hind leg and converting the animals own limb into a peg leg. Recordings of muscle activities and leg movements before and after denervation showed that: (1) the peg leg is actively used in walking and regular bursts occur in motoneurons to leg extensor muscles; (2) driving of motoneuron activity is sufficient to produce fictive bursting in a muscle whose tendon (apodeme) is cut in the ablation; and (3) similar motoneuron activities are found in walking on an oiled glass surface, when the effects of body weight and mechanical coupling are minimized. When distal segments were completely severed in these preparations, leg use and muscle bursting were disrupted but could be restored if the stumps were pressed against the substrate. These results support the hypothesis that feedback from receptors in proximal leg segments indicating forces allows for active leg use in walking.     

The mechanism of ran import into the nucleus by nuclear transport factor 2

The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.