Cellular organization of normal mouse liver: a histological,quantitative immunocytochemical,and fine structural analysis

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.     

A Highlights from MBoC Selection: α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein–coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.     

Muscle aging and oxidative stress in wild-caught shrews

Red-toothed shrews (Soricidae, subfamily Soricinae) are an intriguing model system to examine the free-radical theory of aging in wild mammals, given their short (< 18 months) lifespan and high mass-specific metabolic rates. As muscle performance underlies both foraging ability and predator avoidance, any age-related decline should be detrimental to fitness and survival. Muscle samples of water shrews (Sorex palustris) and sympatrically distributed short-tailed shrews (Blarina brevicauda) were therefore assessed for oxidative stress markers, protective antioxidant enzymes and apoptosis. Activity levels of catalase and glutathione peroxidase increased with age in both species. Similarly, Cu,Zn-superoxide dismutase isoform content was elevated significantly in older animals of both species (increases of 60% in the water shrew, 25% in the short-tailed shrew). Only one oxidative stress marker (lipid peroxidation) was age-elevated; the others were stable or declined (4-hydroxynonenal adducts and dihydroethidium oxidation). Glutathione peroxidase activity was significantly higher in the short-tailed shrew, while catalase activity was 2× higher in water shrews. Oxidative stress indicators were on average higher in short-tailed shrews. Apoptosis occurred in < 1% of myocytes examined, and did not increase with age. Within the constraints of the sample size we found evidence of protection against elevated oxidative stress in wild-caught shrews.     

Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of postsurgical visceral pain in rats

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.     

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.     

In vitro long-term performance study of a near-infrared fluorescence affinity sensor for glucose monitoring

The long-term in vitro performance of a fluorescence affinity sensor for transdermal blood glucose monitoring was investigated. Affinity binding of fluorescently labeled concanavalin A (ConA) was used in this application, as previously described by Ballerstadt and Schultz [Anal. Chem. 17 (2000) 4185-4192). In this paper, the fluorescence emission of the sensor was extended to the near infrared (670 nm) using Alexa647 as the fluorochrome conjugated to concanavalin A. Sensors were alternately exposed to glucose solutions having concentrations of 2.5 and 20 mM with a dwell time of 3 h. The optical output of the sensors was monitored over a 4-month period. The sensors showed an initial increase in fluorescence over the first 3-4 weeks before gradually decreasing, with an approximately linear drop of 25% per month. In order to understand the reasons for the decrease in fluorescence output, further experiments were conducted, including time-dependent membrane leakage tests, solubility tests of ConA, temperature-dependent activity tests of ConA, and fluorescence photo-bleaching tests. From these results, it became evident that the decrease in fluorescence was not due to denaturation of the ConA. The most likely cause was leakage of the fluorescently labeled ConA through the interface between the outer sealant and the membrane. This problem is considered to be solvable and future publications will address this issue. Extrapolation of the experimental data suggests that a leak-proof sensor would be remarkably stable with a fluorescence decrease of only 15% over a 1-year period.     

Sensory processing by neural circuits in Caenorhabditis elegans

The anatomical and developmental constancy of Caenorhabditis elegans belies the complexity of its numerically small nervous system. Indeed, there is an increased appreciation of C. elegans as an organism to study systems level questions. Many recent studies focus on the circuits that control locomotion, egg-laying, and male mating behaviors and their modulation by multiple sensory stimuli.