The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

A fluorescence-based protocol for quantifying angiotensin-converting enzyme activity

The determination of angiotensin-converting enzyme (ACE) activity represents a useful tool in the study of different health pathologies, such as hypertension. This protocol describes a fluorescent assay for measuring ACE activity in vitro with high precision and sensitivity. The method relies on the ability of ACE to hydrolyse the internally quenched fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline. The generation of the fluorescent product o-aminobenzoylglycine can be continuously monitored, preferably using a microtiter-plate fluorometer, though the use of a conventional cuvette fluorometer would also be possible. The method has important advantages with respect to other assays, because it involves only a one-step reagent, is easy to carry out and allows the analysis of an elevated number of samples in shorter times. It can be completed in one and a half hours. In addition, the fact that all reagents are commercially available allows the rapid introduction of the assay into the laboratory.     

Paramyosin content and thick filament structure in insect muscles

Summary The myosin filaments of the flight muscles of the locust Locusta migratoria, the cockchafer Melolontha melolontha and the femur muscles of L. migratoria have solid centers. Those of the flight muscles of the housefly Musca domestica and Drosophila melanogaster are tubular. Electron micrographs of myofibrils of the fleshfly Phormia terrae-novae contain both filament types within one sarcomere and suggest the existence of 4 cross-bridges per crown.Estimates of the ratios of myosin to paramyosin and of myosin to actin on sodium dodecyl sulphate-polyacrylamide gels yielded paramyosin contents of 9% of the thick filament mass for the solid and 2.6% for the tubular filaments (3.8% for P. terrae-novae). Based on the myosinactin ratios up to 6 myosin dimers per crown could be calculated.The molar ratio of actin to arthrin on SDS gels was found to be 3.37 for native and extracted myofibrils of flight muscles from P. terrae-novae. Arthrin is also present in isolated actin filaments suggesting that it is localized in or on the thin filaments. If we assume that it is constituent part of the helices of the thin filaments the number of myosin dimers per crown can be diminished to 4.5, considerably closer to the values obtained by evaluation of electron micrographs.Dedicated to Prof. Dr. Bernhard Rensch on his 85^th birthday     

Mechanical models for insect locomotion: active muscles and energy losses

 We extend the analysis of simple, energy-conserving models for the dynamics of insect locomotion in the horizontal plane developed in Schmitt and Holmes (2000a,b, 2001), where gaits characteristic of steady cockroach running and turning were evoked. In this paper, we include dissipation and energy inputs via active “muscles” in three forms: via prescribed torques at the “hip” pivot, via an active spring element of variable length, and via a pair of Hill-type muscle models representing an extensor/flexor system. Due to mechanical feedback of passive elastic forces, the stable gaits of the conservative models are preserved, and now energy input and absorption balances to additionally stabilize a preferred speed, with only modest neural sensing and feedback being required. However, these bipedal models still cannot simultaneously match observed moment-yaw magnitudes and fore-aft dynamics. Received: 17 September 2001 / Accepted: 20 February 2003 / Published online: 20 May 2003 Correspondence to: P. Holmes (e-mail: pholmes@math.Princeton.EDU) Acknowledgements. This work was supported by DARPA/ONR: N00014-98-1-0747 and DoE: DE-FG02-95ER25238. John Schmitt was partially supported by a DoD Graduate Fellowship, a Wu Fellowship of the School of Engineering and Applied Science, and a George Van Ness Lothrop Honorific Fellowship of the Graduate School at Princeton University. We thank Kenneth Meijer for allowing us to use his muscle model in Sect. 4 and Bob Full and Dan Koditschek for numerous helpful suggestions.     

Prospecting for cellulolytic activity in insect digestive fluids

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-β-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.     

Actin isoforms of insect muscles: Two-dimensional electrophoresis studies

1. Two-dimensional electrophoresis was carried out to determine the isoelectric points of actins from thoracic and leg muscles of various kinds of insects (Coleoptera, Orthoptera, Odonata, Hemiptera, Lepidoptera, Hymenoptera and Diptera).2. The isoelectric points of insect muscle actin ranged from 5.6 to 5.9, appreciably more basic than rabbit skeletal muscle actin (α-actin).3. There was usually one predominant isoform of insect muscle actin except that two isoforms were detected in fly and wasp thoracic muscles.4. In a cicada, the isoelectric points of actins from thoracic, leg and sound organ were 5.65, 5.68 and 5.57, respectively.5. Actins from spider thoracic muscle, and crab claw and crayfish claw muscles also showed the isoelectric points of 5.5–5.7.6. Thus arthropod muscle actins appear to have more basic isoelectric points than vertebrate skeletal muscle ones.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

The three-dimensional structure of the Z disc in insect supercontracting muscles

Two types of Z disc structure have been reported in insect supercontracting muscle fibres: (i) a perforated Z disc where Z material forms a reticulum and (ii) a fragmented Z disc composed of separate, discrete Z bodies. The use of thick (I μm) sections in conjunction with high voltage electron microscopy can distinguish between these two types while conventional thin sections may lead to misinterpretation of structure. It is shown that in one insect, the crane-fly Tipula, the larval body-wall muscles, for which a fragmented Z disc has been proposed, do in fact have a perforated disc. In the wax moth Galleria, homologous muscle fibres have a similar type of Z disc, a finding which indicates the need for re-examination of other lepidopteran muscles claimed to have fragmented discs. A redefinition of supercontraction is proposed which includes reference to the perforated type of Z disc.     

Vibration activity of muscles

Experimental research of autooscillations of biomechanical links in the equilibrium state was carried out. It is found that the induced vibrations of all the links of biomechanical systems can be described by the equations being true for mechanical oscillator vibrations. With the muscular strain being constant and moment inertia of the biomechanical system changing the energy of mechanical vibrations is constant, but the ratio of kinetic energy of the biomechanical oscillator to oscillation frequency is invariant under the muscular strain changes.     

Hydrogel-based protein array for quantifying epidermal growth factor receptor activity in cell lysates

Epidermal growth factor receptor (EGFR) signaling plays an important role in a majority of solid tumors, and therapeutics targeted against EGFR has demonstrated promise in slowing growth of these tumors. However, many of these drugs either have failed to reproduce promising preclinical model results in clinical settings or have been successful in only a subgroup of cancer patients due partly to incomplete assessment of EGFR status in cancer. A patient-customized, predictive diagnostic for the effects of specific anti-EGFR therapies may improve outcomes. Here we report the development of a hydrogel-based protein array for quantitative and reproducible determination of the activity of EGFR directly from cellular extracts. In this study, we used glutathione S-transferase-fused Eps15 (GST–Eps15) fusion proteins immobilized within a polyacrylamide hydrogel as a substrate for quantifying EGFR kinase activity from the extracts of EGFR-expressing cell lines. Significant EGFR up-regulation was detected in a mixture containing 7% EGFR-overexpressing cell lysate diluted in lysate from a cell line expressing low levels of EGFR. In addition, the GST–Eps15 protein array was capable of detecting inhibition of EGFR activity when incubated with different tyrosine kinase inhibitors. These findings establish the potential of this protein–acrylamide copolymer hydrogel array not only to evaluate EGFR status in cancer cell lysates but also to screen for the most promising therapeutics for individual patients and monitor treatment progression.     

Failure to prevent degeneration of insect muscles with pepstatin.

Minimal O₂ consumption (MOC), was elevated during pregnancy, lactation and after cold-acclimation. Since the MOC remained elevated in pregnant rats after removal of the gravid reproductive organs and fetuses, it was concluded that maternal tissues were hypermetabolic. Results of experiments using endocrine ablation (thyroidectomy) or replacement (thyroid hormones) techniques could not resolved the question of whether the thyroid was required to sustain this hypermetabolism.In order to determine whether the increased MOC during lactation was secondary to the calorigenic requirements of milk biosynthesis, the MOC was measured after removal of mammary tissue of the lactating rat. Despite the absence of lactating mammary tissue, the MOC remained elevated.Thyroidectomized rats were successfully acclimated to cold but their MOC did not change. In contrast, intact rats acclimated to cold in the same manner became better cold-acclimated, as judged by survival at colder temperatures, and their MOC was elevated. Cold-acclimation appears to consist of two components, only one of which depends on the presence of an intact thyroid gland. The presence of both components confers maximal tolerance to cold.     

Development of catabolic pathways in insect flight muscles. A comparative study

Activities of enzymes representative of glycolytic and β-oxidative pathways and citric acid and glycerophosphate cycles were measured in the developing flight muscles of three species: Calliphora erythrocephala, Locusta migratoria, and Philosamia cynthia. The activities were measured in vitro under optimal conditions.The enzyme pattern of young flight muscles is quite different from the adult pattern. In the second half of the developmental period final differentiation towards the adult metabolic pattern takes place, in Calliphora leading to exclusively carbohydrate-oxidizing capacities, in Locusta to properties enabling both aerobic glycolytic and β-oxidative processes, whereas Philosamia becomes oriented to fatty acid oxidation. This differentiation starts after a temporary rise of lactate dehydrogenase activity, a phenomenon that seems to be connected with invagination of tracheoblasts into the muscle fibres. This tracheolization might be necessary for differentiation towards the species specific metabolic properties of the adult flight muscle.Theoretical aspects of the enzyme activities, as they were measured in the in vitro assays, are discussed and related to the physiological qualities of the flight muscles of the three species investigated.     

Functional anatomy of vagina muscles in the blood-feeding insect,Rhodnius prolixus

The physiology of the muscles associated with the vagina in the blood-feeding insect, Rhodnius prolixus Stal, was investigated with the use of Methylene Blue staining to visualize the anatomy, and a micro force transducer to record spontaneous and neurally-evoked contractions. The vagina is associated with a dorsal muscle and a set of paired lateral muscles. The dorsal muscle extends from the base of the common oviduct to apodemes located laterally on sternite VIII, the first genital segment. The lateral muscles extend from a medially-located apodeme on the posterior edge of sternite VI around each side of the common oviduct to travel posteriorly along the side of the vagina before inserting laterally on apodemes on sternite VIII. The vagina muscles display spontaneous and neurally-evoked contractions that are prolonged but transient. The response to evoked contractions shows that the muscles are innervated by both excitatory and inhibitory motor axons. The degree of tension generated by evoked contractions is dependent on the frequency of stimulation with maximal tension being generated at 20–30 Hz. This tension, which often exceeds 400 mg, is transient and returns to a baseline within 1 to 2 min during continuous stimulation. These results, which are the first to describe this chamber in this well-studied insect, are discussed with respect to the act of egg laying.     

Calculated background elimination in quantifying nitric-oxide synthase enzyme activity.

Measuring nitric-oxide synthase (NOS) activity by monitoring the conversion of L-arginine to L-citrulline is currently the standard assay for NOS activity. We describe a simple method of quantifying low values of NOS activity by removing the background mathematically. When performing NOS activity studies in samples with low protein amount (< 25 microg/microl), we encountered the problem of sample values that can hardly be differentiated from blank values probably originating from radioactive-labeled arginine in the final eluate. Our method determines mathematically these background values and may be an improvement of the citrulline assay.