Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

Local grassland restoration affects insect communities

1. It is hypothesised that ecological restoration in grasslands can induce an alternative stable state shift in vegetation. The change in vegetation influences insect community assemblages and allows for greater functional redundancy in pollination and refuge for native insect species. 2. Insect community assemblages at eight coastal California grassland sites were evaluated. Half of these sites had undergone restoration through active revegetation of native grassland flora and half were non‐restored. Insects were collected from Lupinus bicolor (Fabaceae) within 2 × 2‐m² plots in spring 2017. Lupinus bicolor is a common native species that is used in California restoration projects, and home and state landscaping projects. 3. Ordination demonstrated that insect community assemblages were different between restored and non‐restored sites. These differences were seen in insect functional groups as well as taxa‐specific differences and were found to be driven by environmental characteristics such as non‐native forb cover. 4. Functional redundancy of herbivores decreased at restored sites, while pollinators became more redundant compared with non‐restored sites. The assemblages of the common species found at restoration sites contained more native insects than those found at non‐restored sites, including species such as Bombus vosnesenskii. 5. Local grassland restoration has the potential to induce an alternative stable state change and affect insect community assemblages. Additionally, it was found that grassland restoration can be a potential conservation tool to provide refugia for bumblebees (Bombus), but additional studies are required to fully understand its broader applicability.     

Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.     

Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.     

The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.     

Enhanced ability of skeletal muscle containing cyclocreatine phosphate to sustain ATP levels during ischemia following beta-adrenergic stimulation

Breast muscle of young chicks fed chow diets containing the creatine analog 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine) accumulated up to 40 mumol/g wet weight of the synthetic phosphagen 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P2-). ATP levels were sustained at high values substantially longer in breast muscle of cyclocreatine-fed chicks, compared to control-fed chicks, during total ischemia initiated 2 h after injection of both groups with the beta-adrenergic agonist isoproterenol (5 mg/kg subcutaneous). For example, in chicks fed 0.5% cyclocreatine for 10-19 days ATP levels in isoproterenol-stimulated breast muscles after 1 h of ischemia at 37 degrees C were 6.1 mumol/g, compared to 1.9 mumol/g for the control-fed group, and after 2 h of ischemia were 3.5 mumol/g compared to 0.6 mumol/g for controls. Creatine-P reserves in isoproterenol-stimulated breast muscles of all dietary groups were essentially exhausted within the first hour of ischemia. In contrast, breast muscle of chicks fed either 1 or 0.5% cyclocreatine still contained 28 and 19 mumol/g of cyclocreatine-P, respectively, after 1 h of ischemia; after 2 h of ischemia, the respective cyclocreatine-P values were 20 and 13 mumol/g. Isoproterenol-stimulated chick breast muscle provides the first skeletal muscle model system for studying the molecular mechanisms by which dietary cyclocreatine helps sustain ATP levels during ischemia. Although adaptive factors are also involved, it is suggested that a significant portion of the ATP-sustaining activity of dietary cyclocreatine in ischemic breast muscle can be attributed to the unique thermodynamic properties of the accumulated cyclocreatine-P. These properties enable cyclocreatine-P to continue to thermodynamically buffer the adenylate system and transport high energy phosphate throughout the long muscle fibers at cytosolic pH values and phosphorylation potentials well below the range where the creatine-P system can function effectively. Synergism between glycolysis and this long-acting synthetic phosphagen might well help delay depletion of ATP levels in skeletal muscles during ischemia. Cyclocreatine feeding provides a unique experimental tool for quantitative evaluation of the proposed protective role of ATP against irreversible cellular damage in skeletal and cardiac muscles during ischemic episodes.     

Messy eaters: Swabbing prey DNA from the exterior of inconspicuous predators when foraging cannot be observed

Complex coevolutionary relationships among competitors, predators, and prey have shaped taxa diversity, life history strategies, and even the avian migratory patterns we see today. Consequently, accurate documentation of prey selection is often critical for understanding these ecological and evolutionary processes. Conventional diet study methods lack the ability to document the diet of inconspicuous or difficult‐to‐study predators, such as those with large home ranges and those that move vast distances over short amounts of time, leaving gaps in our knowledge of trophic interactions in many systems. Migratory raptors represent one such group of predators where detailed diet studies have been logistically challenging. To address knowledge gaps in the foraging ecology of migrant raptors and provide a broadly applicable tool for the study of enigmatic predators, we developed a minimally invasive method to collect dietary information by swabbing beaks and talons of raptors to collect trace prey DNA. Using previously published COI primers, we were able to isolate and reference gene sequences in an open‐access barcode database to identify prey to species. This method creates a novel avenue to use trace molecular evidence to study prey selection of migrating raptors and will ultimately lead to a better understanding of raptor migration ecology. In addition, this technique has broad applicability and can be used with any wildlife species where even trace amounts of prey debris remain on the exterior of the predator after feeding.