Quantifying pteridines in the heads of blow flies (Diptera: Calliphoridae): Application for forensic entomology.

In forensic cases involving entomological evidence, establishing the postcolonization interval (post-CI) is a critical component of the investigation. Traditional methods of estimating the post-CI rely on estimating the age of immature blow flies (Diptera: Calliphoridae) collected from remains. However, in cases of delayed discovery (e.g., when remains are located indoors), these insects may have completed their development and be present in the environment as adults. Adult fly collections are often ignored in cases of advanced decomposition because of a presumed little relevance to the investigation; herein we present information on how these insects can be of value. In this study we applied an age-grading technique to estimate the age of adults of Chrysomya megacephala (Fabricius), Cochliomyia macellaria (Fabricius), and Phormia regina (Meigen), based on the temperature-dependent accumulation of pteridines in the compound eyes, when reared at temperatures ranging from 5 to 35°C. Age could be estimated for all species*sex*rearing temperature combinations (mean r2±SE: 0.90±0.01) for all but P. regina reared at 5.4°C. These models can be used to increase the precision of post-CI estimates for remains found indoors, and the high r2 values of 22 of the 24 regression equations indicates that this is a valid method for estimating the age of adult blow flies at temperatures ≥15°C.

Decomposition of Concealed and Exposed Porcine Remains in the North Carolina Piedmont.

We examined the decomposition and subsequent insect colonization of small pig carrion (Sus scrofa (L.)) placed in concealed and open environments during spring, summer, and fall in Raleigh, North Carolina, as a model for juvenile human remains. Remains were concealed in simulated attics in three manners, ranging from minimal to well-concealed. Concealment had a significant effect on the insect community colonizing the remains across all three seasons; the beetles Necrobia rufipes (DeGeer) (Cleridae) and Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae) were the only species indicative of remains located indoors, whereas numerous fly (Diptera: Calliphoridae, Muscidae, Sepsidae, and Piophilidae) and beetle (Coleoptera: Silphidae, Staphylinidae, and Histeridae) species and an ant species (Hymenoptera: Formicidae, Prenolepis sp.) were indicative of remains located outdoors. Season also significantly affected the insect species, particularly the blow flies (Diptera: Calliphoridae) colonizing remains: Lucilia illustris (Meigen) was indicative of the spring, Cochliomyia macellaria (F.) and Chrysomya megacephala (F.) were indicative of the summer, and Calliphora vicina Robineau-Desvoidy and Calliphora vomitoria (L.) were indicative of the fall. In addition, across all seasons, colonization was delayed by 35­768 h, depending on the degree of concealment. These differences among the insect communities across seasons and concealment treatments, and the effects of concealment on colonization indicate that such information is important and should to be considered when analyzing entomological evidence for criminal investigations.

Blow flies (Diptera: Calliphoridae) survive burial: Evidence of ascending vertical dispersal.

This study was undertaken to determine if immature blow flies could complete development following burial and emerge from the soil as adults. Two species of blow flies, Cochliomyia macellaria and Protophormia terraenovae, were placed at three depths and at three different life stages, in a simulated burial to evaluate the impact of soil on ascending vertical dispersal and fly survival. In soil columns, immature stages of each species were covered with 5, 25 and 50cm of soil. Emerging adult flies of both species reached the surface from all depths at all three immature stages (2nd instar, 3rd instar and pupae). At the 50-cm depth, flies were least successful in reaching the surface when buried as pupae and most successful as late 3rd instar larvae (prepupae). Collectively, more adult flies emerged from the soil if buried as 3rd instars (79.6%) than either 2nd instars or pupae (59.6% and 59.3%, respectively (F(2,159)=14.76, P<0.0001)). Similarly, at shallow burial depths of 5 and 25cm, 75.6% and 70.4% of the adults successfully reached the surface, compared with 52.6% at the 50-cm depth (F(2,159)=15.95, P<0.0001). Second instars demonstrated ascending vertical dispersal behaviours in the soil column by pupating closer to the surface. Nearly half (46.6%) of the C. macellaria 2nd instars buried in 25cm of soil pupated nearer to the surface. Similarly, 45.4% of the P. terraenovae 2nd instars pupated nearer to the surface. When buried at 50cm, approximately 25% of 2nd instars of both species pupated nearer to the surface. When 3rd instars of C. macellaria and P. terraenovae were buried at 120cm, 40% and 4.3% of the adults, respectively, successfully reached the soil surface.

An efficient two-photon-generated photoacid applied to positive-tone 3D microfabrication.

A two-photon-activatable photoacid generator, based on a bis[(diarylamino) styryl]benzene core with covalently attached sulfonium moieties, has been synthesized. The photoacid generator has both a large two-photon absorption cross section (delta = 690 x 10(-50) centimeter(4) second per photon) and a high quantum yield for the photochemical generation of acid (phiH+ = 0.5). Under near-infrared laser irradiation, the molecule produces acid after two-photon excitation and initiates the polymerization of epoxides at an incident intensity that is one to two orders of magnitude lower than that needed for conventional ultraviolet-sensitive initiators. This photoacid generator was used in conjunction with a positive-tone chemically amplified resist for the fabrication of a three-dimensional (3D) microchannel structure.

Channel behavior in a gamma-aminobutyrate transporter.

Current produced by a gamma-aminobutyrate (GABA) transporter stably transfected into a mammalian cell line was observed in cell-attached and excised membrane patches. When GABA was absent, a fraction of the transporters produced cation-permeable channels. When GABA plus Na+ was on either side of the membrane, the majority of transporters produced a high-frequency current noise attributed to the movement of ions in an occluded pore.

A GABA transporter operates asymmetrically and with variable stoichiometry.

Membrane currents produced by the expression of a rat GABA transporter (GAT-1) stably transfected into HEK293 cells were characterized with a whole-cell voltage clamp. Three modes of function were identified: ex-gated currents produced by extracellular GABA, in-gated currents produced by intracellular GABA, and uncoupled currents produced in the absence of GABA. The ex-gated current was not the reversal of the in-gated current; moreover, the stoichiometry between GABA and co-ions was not always fixed. Each mode of function required a different set of ions on the two sides of the membrane. We made rapid solution changes and observed an allosteric effect of Na+ that only occurred at the extracellular surface. Thus, the GAT-1 transporter does not behave like a recirculating carrier but may be described as a pore with ion gates at either end that are controlled in part by allosteric sites.

Ions required for the electrogenic transport of GABA by horizontal cells of the catfish retina.

1. Solitary horizontal cells were isolated from catfish retinas. Membrane currents activated by extracellular and intracellular GABA were characterized during a whole-cell voltage clamp. 2. Extracellular GABA activated two currents: a GABAA current, and an 'influx' current mediated by a GABA transporter. The influx current was studied after the GABAA current was blocked with 0.5 mM picrotoxin. The influx current required extracellular Na+ and Cl-. Extracellular Na+ could not be replaced by another alkali metal cation. 3. The influx current also depended upon the identity of ions in the intracellular solution. Either an intracellular alkali metal cation or Cl- was required to produce an influx current. 4. The influx current was inward at -75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the current depended upon the ion composition of the intracellular solution and could be inward, zero or outward. 5. The introduction of GABA into a cell during the course of an experiment produced an outward current. This 'efflux' current was small at -75 mV and increased with depolarization. The efflux current required intracellular Na+ and Cl-. Intracellular Na+ could not be replaced by another alkali metal cation. 6. The efflux current also depended upon the identity of ions in the extracellular solution. An extracellular alkali metal cation was required to produce an efflux current. Removing extracellular Cl- did not affect the efflux current. 7. The outward movement of GABA produced a local accumulation in extracellular GABA concentration that could be detected by the activation of the GABAA current. GABA efflux only occurred during conditions that produced an efflux current. Electroneutral efflux did not occur. 8. In the absence of GABA, extracellular alkali metal cations produced a 'leakage' current. The leakage current was inward at -75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the leakage current depended, like the GABA influx current, upon the ion composition of the intracellular solution and could be inward, zero or outward. The addition of GABA to the intracellular solution produced an efflux current and suppressed the leakage current. 9. We conclude that the transporter mediates electrogenic influx, efflux and leakage. Each mode of operation depends upon ions on both sides of the membrane. Influx and efflux are not symmetrical.

Bilateral single ectopic ureters: diagnosis using transrectal ultrasound.

An unusual case of bilateral, single ectopic ureters inserting into the prostate gland presenting with hydronephrosis and renal insufficiency is reported. The diagnosis was made by transrectal and abdominal ultrasound.

Electrochemical monitoring of brain ascorbic acid changes associated with hypoxia, spreading depression, and seizure activity.

In vivo electrochemistry has been a valuable tool in detecting real time neurochemical changes in extracellular fluid. Absolute selectivity has been difficult to achieve previously, but we report here a carbon fiber electrode and measurement technique which is specific for one oxidizable species: ascorbic acid. Ascorbic acid is highly concentrated in extra- as well as intracellular brain spaces, and appears to undergo dynamic changes in response to a variety of physiological and pathophysiological circumstances. Recent studies have implicated glutamatergic mechanisms which give rise to extracellular changes in brain ascorbate, and we confirm and extend these observations. Preliminary studies, directed towards examining ascorbic acid as an index and/or result of hypoxia, spreading depression, and seizure activity, have been undertaken and the results are reported herein.

Anoxia-resistant turtle brain maintains ascorbic acid content in vitro.

The isolated turtle brain maintains intra- and extracellular concentrations of ascorbate when incubated in ascorbate-free physiological saline for as long as 24 h. After incubation for 1 h, total tissue content of ascorbate in the turtle cerebellum was the same as in unincubated controls. After 20-24 h, tissue ascorbate content remained at 65% of control levels, while extracellular ascorbate concentration, measured with carbon fiber voltammetric microelectrodes, was 56% of the initial value. For an intermediate incubation period of 6 h, reduced ascorbate content was maintained at about 80% of control levels, regardless of whether incubation was under normal conditions or in the absence of glucose or oxygen. By contrast, only 4% of the ascorbate content of guinea pig brain slices remained after a 6 h incubation. Maintenance of high levels of ascorbate by the anoxia-resistant turtle brain could be an important factor in the amelioration of oxidative injury in this tissue. Inclusion of ascorbate in media used for in vitro studies of mammalian brain tissue is recommended.

The pharmacological profile of glutamate-evoked ascorbic acid efflux measured by in vivo electrochemistry.

A recently described in vivo voltammetric electrode selectively records rapid changes in extracellular fluid (ECF) levels of ascorbic acid. Using this detector, the nature of glutamate-induced efflux of ascorbate into ECF was investigated using pharmacological tools. Ascorbate signals were shown to be directly related to amounts of microinjected glutamate. Blockers of glutamate reuptake, homocysteic acid and D,L-threo-beta-hydroxy-aspartic acid, virtually eliminate the ascorbate signal. A more specific reuptake blocker (the stilbene isothiocyano derivative (SITS) does not completely inhibit ascorbate efflux, suggesting that the glutamate uptake which is coupled to ascorbic acid exchange is both neuronal and glial in nature. Other pharmacological experiments indicate that excitatory amino acid receptors are not involved in the glutamate-elicited ascorbate efflux; it is primarily a function of the glutamate/ascorbate heteroexchange process as described earlier. The possible role(s) of brain ascorbate in the general functioning of the pervasive glutamate neurotransmitter systems are discussed.

Antioxidants prevent the interaction of 3,4-dihydroxyphenylacetic acid at the dopamine D2 receptor.

Antioxidants such as ascorbic acid (AA) and dithiothreitol (DTT), can prevent 3,4-dihydroxyphenylacetic acid (DOPAC) inhibition of [3H]spiperone binding to neuronal dopamine D2 receptors. The DOPAC quinone produced from auto-oxidation is believed to be responsible for the irreversible inhibition noted. Quinone conjugation to proteins occurs readily in vivo, and thus, auto-oxidation of DOPAC and other endogenous catechol containing compounds could be an important component of protein modification during on-going neuronal physiology.

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples.

The ascorbic acid (AA)-dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.

Dynamic changes in extracellular fluid ascorbic acid monitored by in vivo electrochemistry.

Voltammetric carbon fiber electrodes and a measuring protocol were designed to monitor extracellular changes in rat brain ascorbic acid (AA). Very fast variations of AA (less than 60 s in duration) as well as much slower changes can be followed. Basal and stimulated levels of AA, determined with the enzyme ascorbate oxidase (AAO), confirm the detection is selective for AA. Microinjections of glutamate (Glu) into various brain regions gave rise to rapid electrochemical signals ascribed to the efflux of AA into the extracellular fluid.

Inhibition of luteinizing hormone/human chorionic gonadotropin-stimulable adenylyl cyclase activity in rat luteal membranes by nonsteroidal component(s) in human follicular fluid.

We examined the ability of nonsteroidal components of human follicular fluid (hFF) to alter gonadotropin responsiveness using the LH/hCG-sensitive adenylyl cyclase system of rat luteal membranes. Follicular aspirates were obtained from regularly ovulatory women (n = 10) whose follicles were stimulated by human menopausal gonadotropin and hCG as part of an in vitro fertilization program. hFF from large follicles was pooled and extracted with 10% (wt/vol) activated charcoal. Maximal hCG stimulation of adenylyl cyclase activity obtained with 10 micrograms/ml hCG and 100 microM of the hydrolysis-resistant GTP analog guanyl 5'-yl-imidodiphosphate was significantly inhibited by hFF in a dose-dependent manner. Addition of about 500 micrograms hFF protein caused inhibition of 70% compared to the control value. Fractionations of hFF by ultrafiltration using membranes of precalibrated pore size demonstrated that the inhibitory activity was associated with a less than 10,000 mol wt fraction; 3 micrograms protein/assay of this fraction resulted in 50% inhibition (IC50) of maximal hCG stimulation. The inhibitory activity also passed through an Amicon YM-2 membrane (mol wt retention, 1,000), but not through an Amicon YC-05 membrane (mol wt retention, 500). An IC50 of about 0.01 microgram protein/assay was found for both the 500-1,000 and the 1,000-5,000 mol wt fractions. NaF or forskolin-stimulated adenylyl cyclase activity was not altered by unfractionated hFF or by the 500-10,000 mol wt subfractions, suggesting that inhibition was limited to LH/hCG stimulation. Further analysis of the effects of low mol wt fraction on hCG stimulation of adenylyl cyclase indicated that enzyme inhibition was not accompanied by a shift in the hCG concentration required for half-maximal stimulation (the apparent activation constant) compared to dose-response curves obtained in the absence of added fraction. Equilibrium binding studies showed that [125I]hCG interaction with luteal membranes was significantly inhibited by hFF; 7 micrograms protein/assay of the less than 10,000 mol wt fraction reduced specific binding by 60%. Moreover, kinetic analysis carried out in the absence or presence of a fixed amount of low mol wt fractions revealed a competitive type of binding inhibition. Our data demonstrate that a nonsteroidal component(s) of hFF has a direct inhibitory effect on LH/hCG-responsive luteal adenylyl cyclase and that the inhibitor(s) exerts its actions through a mechanism involving competition with LH/hCG for the same binding sites.

Psychological stress and the passage of a standard meal through the stomach and small intestine in man.

Gastric emptying half-time and mouth to caecum transit time of a solid meal were measured in eight normal volunteers, once during a period of psychological stress and again during a period of relative calm. No consistent or significant effect on gastric emptying was observed, but mouth to caecum transit times were faster in all subjects and this difference was highly significant (p<0.01).

Effect of prolonged exercise on the passage of a solid meal through the stomach and small intestine.

The effect of intermittent moderate exercise on the passage of a solid meal, labelled with radioactive Technetium sulphur colloid, through the stomach and small intestine was investigated by paired studies on seven healthy volunteers. Measurements of gastric radioactivity and breath hydrogen exertion were recorded every 10 minutes while subjects exercised in a controlled manner while seated on a bicycle ergometer. These were compared with values obtained during a separate experiment while the same subjects sat upright in a chair. Exercise significantly accelerated gastric emptying (control t 1/2 = 1.5 +/- 0.1 h; exercise t 1/2 = 1.2 +/- 0.1 h; p less than 0.02) but had no significant effect on small bowel transit time.