An inexpensive method for remotely monitoring nest activity

ABSTRACT.   In studies of avian nest success, investigators often face the difficult task of periodically checking nest status while at the same time limiting observer influence on nest survival. Remotely monitoring nests using temperature data loggers is one method that allows for continuous data capture regarding nest status (i.e., active vs. inactive) without the negative effects associated with repeated nest checks. We used small temperature data loggers (Thermochron iButtons) to remotely monitor nests of Long-billed Curlews ( Numenius americanus ) in northeastern Nevada. Data loggers programmed to record temperature at 10-min and 20-min intervals were placed in curlew nests. Data loggers were set to collect data throughout the nesting cycle to determine onset of incubation and timing of nest failure. On average, Long-billed Curlews began incubating approximately 3 d after the first egg was laid and onset of incubation coincided with the laying of the third egg. iButtons allowed us to determine when incubation was terminated in 17 of 23 unsuccessful Long-billed Curlew nests, including 13 of 17 depredated nests. The presence of iButtons in Long-billed Curlew nests did not affect daily survival rate, egg hatchability or rate of nest abandonment. iButtons are an efficient and practical means for remotely monitoring nests of large egg-laying birds, such as the Long-billed Curlew.     

Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.     

Environmental regulation of mutation rates at specific sites

Recent studies on bacterial adaptation to stress suggest that bacteria can regulate the generation of mutations at specific sites in response to environmental conditions. Here, we review these findings and discuss the circumstances under which these mechanisms might prove advantageous.     

Microchip transponder thermometry for monitoring core body temperature of antelope during capture

Hyperthermia is described as the major cause of morbidity and mortality associated with capture, immobilization and restraint of wild animals. Therefore, accurately determining the core body temperature of wild animals during capture is crucial for monitoring hyperthermia and the efficacy of cooling procedures. We investigated if microchip thermometry can accurately reflect core body temperature changes during capture and cooling interventions in the springbok (Antidorcas marsupialis), a medium-sized antelope. Subcutaneous temperature measured with a temperature-sensitive microchip was a weak predictor of core body temperature measured by temperature-sensitive data loggers in the abdominal cavity (R²=0.32, bias >2 °C). Temperature-sensitive microchips in the gluteus muscle, however, provided an accurate estimate of core body temperature (R²=0.76, bias=0.012 °C). Microchips inserted into muscle therefore provide a convenient and accurate method to measure body temperature continuously in captured antelope, allowing detection of hyperthermia and the efficacy of cooling procedures.     

Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages

The nonsynonymous (amino acid-altering) to synonymous (silent) substitution rate ratio (omega = d(N)/d(S)) provides a measure of natural selection at the protein level, with omega = 1, >1, and <1, indicating neutral evolution, purifying selection, and positive selection, respectively. Previous studies that used this measure to detect positive selection have often taken an approach of pairwise comparison, estimating substitution rates by averaging over all sites in the protein. As most amino acids in a functional protein are under structural and functional constraints and adaptive evolution probably affects only a few sites at a few time points, this approach of averaging rates over sites and over time has little power. Previously, we developed codon-based substitution models that allow the omega ratio to vary either among lineages or among sites. In this paper we extend previous models to allow the omega ratio to vary both among sites and among lineages and implement the new models in the likelihood framework. These models may be useful for identifying positive selection along prespecified lineages that affects only a few sites in the protein. We apply those branch-site models as well as previous branch- and site-specific models to three data sets: the lysozyme genes from primates, the tumor suppressor BRCA1 genes from primates, and the phytochrome (PHY) gene family in angiosperms. Positive selection is detected in the lysozyme and BRCA genes by both the new and the old models. However, only the new models detected positive selection acting on lineages after gene duplication in the PHY gene family. Additional tests on several data sets suggest that the new models may be useful in detecting positive selection after gene duplication in gene family evolution.     

An assay specific for the active form of liver phosphorylase kinase

An assay specific for the active form of liver phosphorylase kinase (EC 2.7.1.38) has been developed utilizing inhibition of the inactive form of phosphorylase kinase by β-glycerophos, phate and ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid. Following in vitro activation the results compared favorably with those obtained using a less specific assay previously available. (J. R. Vandenheede, S. Keppens, and H. DeWulf, 1977, Biochim. Biophys. Acta.481, 463–470;D. D. Doorneweerd, A. W. H. Tan, and F. Q. Nuttall, 1978, Diabetes27, 474). The in vitro activation of phosphorylase kinase was not associated with the formation of a small-molecular-weight form of the enzyme. The utility of the assay in monitoring in vivo interconversion reactions in response to various physiological stimuli was demonstrated.     

Ionizable side chains at catalytic active sites of enzymes

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å³. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.     

An inexpensive camera system for monitoring cavity nests

ABSTRACT A variety of pole‐mounted cameras have been developed for monitoring nest cavities. However, currently available camera systems may either be prohibitively expensive or difficult to assemble. I developed an inexpensive (<$500 US) and easily assembled camera system that allows researchers to monitor cavity nests from the ground. The system consists of a small camera, a cable connecting the camera to a ground‐level power source and laptop computer, and a flexible neck connecting the camera to a telescoping pole. During a study of Red‐headed Woodpeckers (Melanerpes erythrocephalus), I used this camera to inspect 16 nests and found that the images were clear and allowed accurate counts of eggs and nestlings. This camera system uses standard, off‐the‐shelf components, and can easily be altered. The design is not appropriate for humid or dense‐canopy environments because of the inclusion of a laptop and its wired design. However, this design makes the system inexpensive and allows researchers to save, edit, and view nest inspection recordings.     

Monitoring of cellular senescence by DNA-methylation at specific CpG sites

Replicative senescence has fundamental implications on cell morphology, proliferation, and differentiation potential. Here, we describe a simple method to track long-term culture based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic senescence signature can be used as biomarker for various cell types to predict the state of cellular senescence with regard to the number of passages, population doublings, or days of in vitro culture.     

Evidence for essential arginine residues at the active sites of maize branching enzymes

Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues. Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis. BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5. The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics. Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO. BE inactivation was positively correlated with [¹⁴C]PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.Abbreviations BE branching enzyme - BCA bicinchoninic acid - BSA bovine serum albumin - Glc-1-P glucose-1-phosphate - IPTG isopropyl-d-thiogalactoside - PGO phenylglyoxal - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium docecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - TEA triethanolamine     

Low- and high-resolution mapping of DNA damage at specific sites

Measurement of DNA damage and repair at the nucleotide level in intact cells has provided compelling evidence for the molecular details of these events as they occur in intact organisms. Furthermore, these measurements give the most accurate picture of the rates of repair in different structural domains of DNA in chromatin. In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolution in yeast cells. The low-resolution method couples damage-specific strand breaks in DNA with indirect end-labeling to measure DNA lesions over a span of 1.5 to 2 kb of DNA sequence. The resolution of this method is limited by the resolution of DNA length measurements on alkaline agarose gels (about +/-20 bp on average). The high-resolution method uses streptavidin magnetic beads and special biotinylated oligonucleotides to facilitate end-labeling of DNA fragments specifically cleaved at damage sites. The latter method maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequencing gels. These methods are used in tandem for answering questions regarding DNA damage and repair in different chromatin domains and states of gene expression.     

Performance of stationary and portable passive transponder detection systems for monitoring of fish movements

A stationary system for long-range detection of PIT tags in fish was efficient under high water conditions in streams. A portable system was particularly effective for detecting habitat use by fish without recapture.     

A system for monitoring and improving animal visibility and its implications for zoological parks

With the growing trend in zoos to build complex, naturalistic exhibits comes the potential for exhibits to be so densely vegetated or complex that animals are not easily seen by zoo visitors. This can negatively impact the visitor's visiting experience and the zoo's ability to communicate conservation and education messages. Over the past 9 years, Disney's Animal Kingdom^® has developed a process for monitoring and improving the visibility of animals on display to the public. This animal visibility process utilizes a data collection system whereby systematic observations are collected each week. The percentage of observations where at least one animal was visible is recorded for each species and compared to an 80% visibility criterion. Species that do not reach this criterion for 4 consecutive weeks are discussed at animal management meetings. If the problems associated with animal visibility cannot be easily solved, the animal‐care teams partner with the research team to conduct a second process, called the Visibility Issues Process. This process provides additional information for the animal‐care team to utilize in developing a plan to improve visibility. Although the processes described here are specific to the infrastructure at Disney's Animal Kingdom^®, the basic concepts of (1) a formalized visibility data collection process, (2) a visibility criterion to which managers of species are held accountable, and (3) a process for planning to improve animal visibility without negatively impacting animal welfare are fundamental concepts that can be developed at individual institutions and incorporated into that zoo's existing infrastructure. Zoo Biol 29:68–79, 2010. © 2009 Wiley‐Liss, Inc.     

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.