Role of the tubulin-microtubule system in lymphocyte activation

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.     

Effectiveness of Partial Sedation to Reduce Stress in Captured Mule Deer

Information garnered from the capture and handling of free-ranging animals helps advance understanding of wildlife ecology and can aid in decisions on wildlife management. Unfortunately, animals may experience increased levels of stress, injuries, and death resulting from captures (e.g., exertional myopathy, trauma). Partial sedation is a technique proposed to alleviate stress in animals during capture, yet efficacy of partial sedation for reducing stress and promoting survival post-capture remains unclear. We evaluated the effects of partial sedation on physiological, biochemical, and behavioral indicators of acute stress and probability of survival post-capture for mule deer (Odocoileus hemionus) that were captured via helicopter net-gunning in the eastern Greater Yellowstone Ecosystem, Wyoming, USA. We administered 10–30 mg of midazolam and 15 mg of azaperone intramuscularly (IM) to 32 mule deer in 2016 and 53 mule deer in 2017, and maintained a control group (captured but not sedated) of 38 mule deer in 2016 and 54 mule deer in 2017. To evaluate indicators of acute stress, we measured heart rate, blood-oxygen saturation, body temperature, respiration rate, and levels of serum cortisol. We recorded number of kicks and vocalizations of deer during handling and evaluated behavior during release. We also measured levels of fecal glucocorticoids as an indicator of baseline stress. Midazolam and azaperone did not reduce physiological, biochemical, or behavioral indicators of acute stress or influence probability of survival post-capture. Mule deer that were administered midazolam and azaperone, however, were more likely to hesitate, stumble or fall, and walk during release compared with individuals in the control group, which were more likely to trot, stot, or run without stumbling or falling. Our findings suggest that midazolam (10–30 mg IM) and azaperone (15 mg IM) may not yield physiological or demographic benefits for captured mule deer as previously assumed and may pose adverse effects that can complicate safety for captured animals, including drug-induced lethargy. Although we failed to find efficacy of midazolam and azaperone as a method for reducing stress in captured mule deer, the efficacy of midazolam and azaperone or other combinations of partial sedatives in reducing stress may depend on the dose of tranquilizer, study animal, capture setting, and how stress is defined. © 2020 The Wildlife Society.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

Spatial memory shapes migration and its benefits: evidence from a large herbivore

From fine‐scale foraging to broad‐scale migration, animal movement is shaped by the distribution of resources. There is mounting evidence, however, that learning and memory also guide movement. Although migratory mammals commonly track resource waves, how resource tracking and memory guide long‐distance migration has not been reconciled. We examined these hypotheses using movement data from four populations of migratory mule deer (n = 91). Spatial memory had an extraordinary influence on migration, affecting movement 2–28 times more strongly than tracking spring green‐up or autumn snow depth. Importantly, with only an ability to track resources, simulated deer were unable to recreate empirical migratory routes. In contrast, simulated deer with memory of empirical routes used those routes and obtained higher foraging benefits. For migratory terrestrial mammals, spatial memory provides knowledge of where seasonal ranges and migratory routes exist, whereas resource tracking determines when to beneficially move within those areas.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.     

Distribution and excretion of a phosphorothioate oligonucleotide in rats with experimentally induced renal injury

The effects of renal injury on the urinary excretion and tissue distribution of a 20-mer phosphorothioate oligonucleotide were investigated in male Sprague-Dawley rats. Renal injury was produced by treating the rats with either 5.0 mg/kg cisplatin or 2.5 mg/kg of a monoclonal antibody (mAb) directed toward Thy1.1. Controls received saline. Three days after cisplatin treatment or 2 days after anti- Thy1.1 treatment, the rats received 10 mg/kg ISIS 3521. Blood was collected at various times to assess the plasma concentrations of ISIS 3521, and rats were killed at various times from 6 to 48 hours after intravenous (i.v.) infusion of oligonucleotide to assess tissue concentrations by capillary gel electrophoresis (CGE). Cisplatin and anti-Thy1.1 antibody produced histologic and biochemical changes consistent with proximal tubular damage and glomerular damage, respectively. Urinary excretion of oligonucleotides was increased 2- to 4-fold of control; however, this amount accounted for only 1% to 2% of dose compared to 0.5% in controls. Proximal tubular damage reduced renal accumulations of ISIS 3521 and other oligonucleotide metabolites, but there were no obvious compensatory increases in concentrations in other organs except for a slight increase in spleen levels of total oligonucleotide. Glomerular damage was not associated with any change in oligonucleotide disposition. Immunohistochemical studies showed no evidence of alterations in the pattern of distribution within the injured kidney. The data suggest that acute renal dysfunction, either renal tubular or glomerular, does not markedly alter the urinary elimination and tissue deposition of a phosphorothioate oligonucleotide.     

Biogeographical concordance and efficiency of taxon indicators for establishing conservation priority in a tropical rainforest biota

Prioritizing areas for conservation requires the use of surrogates for assessing overall patterns of biodiversity. Effective surrogates will reflect general biogeographical patterns and the evolutionary processes that have given rise to these and their efficiency is likely to be influenced by several factors, including the spatial scale of species turnover and the overall congruence of the biogeographical history. We examine patterns of surrogacy for insects, snails, one family of plants and vertebrates from rainforests of northeast Queensland, an area characterized by high endemicity and an underlying history of climate-induced vicariance. Nearly all taxa provided some level of prediction of the conservation values for others. However, despite an overall correlation of the patterns of species richness and complementarity, the efficiency of surrogacy was highly asymmetric; snails and insects were strong predictors of conservation priorities for vertebrates, but not vice versa. These results confirm predictions that taxon surrogates can be effective in highly diverse tropical systems where there is a strong history of vicariant biogeography, but also indicate that correlated patterns for species richness and/or complementarity do not guarantee that one taxon will be efficient as a surrogate for another. In our case, the highly diverse and narrowly distributed invertebrates were more efficient as predictors than the less diverse and more broadly distributed vertebrates.     

Simulated brain tumor growth dynamics using a three-dimensional cellular automaton

We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice.     

The effect of variation of the lingual shape of mandibular complete dentures on lingual resistance to lifting forces

Increasing life expectancy, age related reduction in adaptability and progressive severe mandibular resorption all add to the importance of any factor improving the prosthetic success. Objective : To investigate the effect of two different lingual shapes of lower dentures on patients' ability to resist lifting forces. Design : Tongue pressures on the lingual surface of complete mandibular experimental dentures were recorded from mid-line, premolar and molar transducers. Two experimental prostheses were fabricated for each subject: one conventionally contoured, the other formed by piezography. Setting : A clinical research laboratory. Subjects Five experienced complete denture wearers between age 64 and 82 years. Intervention Lifting forces were applied at the midline, left and right premolar sites in random order. Main outcome measures : Peak resistance to lifting forces and lingual pressures used during these tests. Results : Lingual pressures exerted anteriorly were dramatically higher than those on premolar and molar surfaces. Significantly higher pressures were used to resist lifting forces applied to piezographically than conventionally formed contours; correspondingly, significantly higher lifting peak forces were, on average, resisted. Conclusions : Providing a lower denture with a piezographically produced lingual surface was shown, in this preliminary study, to enhance tongue retentive ability over a conventional design. It seems reasonable to maximise retentive potential with oblique sublingual polished surfaces and minimise the adaptive demand, particularly for older patients, by using a piezographic technique which “customises” the contour and precludes over-extension.