Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.     

Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.     

Effects of lung congestion and oleic acid injury on the Hering-Breuer reflex

Breathing and the Hering-Breuer (HB) reflex may be stimulated by congestion and by acute lung injury, but there is disagreement about the effects of both stimuli. This study evaluated these effects using greater stimulus isolation and control of secondary interactions than have previously been employed. Pressurization of lung vessels and left heart and oleic acid injury were individually imposed on anesthetized open-chest dogs perfused with an external pump and gas exchanger. Lungs were inflated in steps before and during those stimuli. The HB reflex was evaluated from graphs of breathing frequency (fr) vs. airway pressure. Congestion itself had no significant sustained effect on fr, but it slightly depressed the HB reflex. Oleic acid tachypnea that was depressed to pretreatment fr by inflation, implying enhancement of the HB response. Capsaicin and oleic acid had similar effects. Vagal cooling to 8 degrees C slightly depressed the effects of oleic acid and capsaicin, had no effect on the sustained fr response to congestion, and reversed the inhibitory effect of inflation. A stimulation of breathing or an enhancement of the HB reflex by congestion was not confirmed, but oleic acid increased fr and the HB reflex.     

A simple,band‐mounted device to measure behaviorally modified thermal microclimates experienced by birds

Birds encounter climate at the scale of microclimates that can vary rapidly in time and space and so understanding potential vulnerability and adaptations to those microclimates requires fine‐scale measurements that accurately track thermal exposures. However, few options exist for recording the microclimates actually experienced by birds (realized microclimates). We constructed and tested a simple, low‐cost, temperature logger for recording the realized microclimates of ground‐nesting birds. We developed attachment protocols for band‐mounting Thermochron iButtons on Ring‐billed Gull (Larus delawarensis) chicks. We tested these mounted, temperature‐logging devices on 20 chicks weighing > 200 g (device weight was 4 g), attaching devices for 48 h and observing behavior before and after attachment and removal. Devices recorded temperature immediately surrounding the leg at 2‐min intervals. Recorded temperatures were strong predictors of observed thermoregulatory behaviors (panting and sitting), outperforming predictions based on air temperatures measured by basic, static approaches. Through comparison with matched controls (chicks with just a band), we detected no adverse physiological effects of devices, no effects on social or feeding behavior, and only a short‐term decrease in inactivity immediately after device attachment (likely due to increased preening). By attaching iButtons to the legs of birds, we quantified realized thermal exposure, integrating air temperature, modes of environmental heat transfer, and bird behavior at microclimatic scales. Although not yet validated for broader use, our approach (including possible miniaturization) should be suitable to measure thermal exposure of adults, not just chicks, allowing collection of data concerning thermal exposures during flight under field conditions. At ~ $25 USD per device, our approach facilitates experimental protocols with robust sample sizes, even for relatively modest budgets.     

Global domination by crazy ants: phylogenomics reveals biogeographical history and invasive species relationships in the genus Nylanderia (Hymenoptera: Formicidae)

Nylanderia (Emery) is one of the world's most diverse ant genera, with 123 described species worldwide and hundreds more undescribed. Fifteen globetrotting or invasive species have widespread distributions and are often encountered outside their native ranges. A molecular approach to understanding the evolutionary history and to revision of Nylanderia taxonomy is needed because historical efforts based on morphology have proven insufficient to define major lineages and delimit species boundaries, especially where adventive species are concerned. To address these problems, we generated the first genus-wide genomic dataset of Nylanderia using ultraconserved elements (UCEs) to resolve the phylogeny of major lineages, determine the age and origin of the genus, and describe global biogeographical patterns. Sampling from seven biogeographical regions revealed a Southeast Asian origin of Nylanderia in the mid-Eocene and four distinct biogeographical clades in the Nearctic, the Neotropics, the Afrotropics/Malagasy region, and Australasia. The Nearctic and Neotropical clades are distantly related, indicating two separate dispersal events to the Americas between the late Oligocene and early Miocene. We also addressed the problem of misidentification that has characterized species-level taxonomy in Nylanderia as a result of limited morphological variation in the worker caste by evaluating the integrity of species boundaries in six of the most widespread Nylanderia species. We sampled across ranges of species in the N. bourbonica complex (N. bourbonica (Forel) + N. vaga (Forel)), the N. fulva complex (N. fulva (Mayr) + N. pubens (Forel)), and the N. guatemalensis complex (N. guatemalensis (Forel) + N. steinheili (Forel)) to clarify their phylogenetic placement. Deep splits within these complexes suggest that some species names – specifically N. bourbonica and N. guatemalensis – each are applied to multiple cryptic species. In exhaustively sampling Nylanderia diversity in the West Indies, a ‘hot spot’ for invasive taxa, we found five adventive species among 22 in the region; many remain morphologically indistinguishable from one another, despite being distantly related. We stress that overcoming the taxonomic impediment through the use of molecular phylogeny and revisionary study is essential for conservation and invasive species management.     

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.