A preliminary investigation into the comparison of dissolution/digestion techniques for the chemical characterization of polyurethane foam.

Due to their widespread use in domestic and commercial premises, polyurethane foams, as either fragmented or bulk foam, are types of evidence commonly found at crime scenes. The traditional approach to determining the evidential value of polyurethane foam (PF) involves comparing recovered and control fragments under low and high magnification, under various lighting conditions, as well as the comparison of their respective dye spectra. As with most forms of trace evidence, chemical comparison is also desirable. In this work, two approaches to chemically comparing foam fragments were investigated, i.e. inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis of the Tin (Sn) content in different foam types; and gas chromatography-flame ionization detection (GC-FID) analysis of soluble components in PFs mobilized by dichloromethane. Seven different foam types were studied and their Sn content was found to be different. They also produced characteristic GC-FID chromatographic profiles whose compounds were identified with gas chromatography-mass spectrometry (GC-MS) analysis. This study suggests that incorporating chemical data obtained from GC-FID/GC-MS and ICP-OES into a case involving PF could be advantageous, as this will enable the forensic scientist to broaden the comparison between control and recovered fragments, and further assess the strength of the evidence. However, ICP-OES analysis is a destructive technique with a relatively short sample turnaround time, whilst GC-FID analysis is more time-consuming and non-destructive, requiring corroboration with GC-MS data. The values of these two analytical techniques in the forensic chemical characterization of PFs are discussed.

Organophosphate inhibitors: the reactions of bis(p-nitrophenyl) methyl phosphate with liver carboxylesterases and alpha-chymotrypsin.

Bis(p-nitrophenyl) methyl phosphate (BNMP) has been tested as a spectrophotometric titrant for a group of serine hydrolases. Bis(p-nitrophenyl) methyl phosphate reacts rapidly with liver carboxylesterases from chicken, sheep, and horse, and more slowly with alpha-chymotrypsin, releasing 2 mol of p-nitrophenol per active site titrated, and producing a phosphorylated enzyme very stable to dephosphorylation. However, pig liver carboxylesterase produces 2.2 mol of p-nitrophenol per active site titratedmreaction of pig and chicken liver carboxylesterases with bis(p-nitrophenyl) [3H]methyl [32P]phosphate clarified this differencemone molecule of the chicken enzyme reacts with one molecule of bis(p-nitrophenyl) methyl phosphate, releasing both p-nitrophenol residues, and resulting in an inhibited enzyme with one phosphorus atom and one methyl group covalently bound. Pig enzyme reacts rapidly, forming (presumably) methyl p-nitrophenyl phosphoryl-carboxylesterasemthis further reacts, concurrently producing methyl phosphoryl-carboxylesterase plus p-nitrophenol, or free enzyme plus methyl p-nitrophenyl phosphate, in the ratio of about 5 : 1 at pH 7.55. The free enzyme produced undergoes further reaction with bis(p-nitrophenyl) methyl phosphate until all the carboxylesterase is inhibited.

Telomerase: anti-cancer target or just a fascinating enzyme?

Telomerase activity is upregulated in most types of malignant tumor. Highly selective small molecule inhibitors will be needed to understand the biological basis for this observation and to determine if telomerase is a viable target for chemotherapy.

Cellular delivery of peptide nucleic acids and inhibition of human telomerase.

BACKGROUND: Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function. Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization. RESULTS: We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly. Relative potencies of inhibition delineate putative structural domains. We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs. CONCLUSIONS: PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents. The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes. Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.

Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits.

Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-11264], and the rod PDE 11 kDa inhibitory subunit in E. coli, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar Ki values (approximately 80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has Ki values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDE gamma.

Hemispheric specialization for skilled perceptual organization by chessmasters.

The right cerebral hemisphere may be relatively specialized for parsing simple visual stimuli according to default rules, such as the Gestalt laws of perceptual organization, whereas the left cerebral hemisphere may be relatively specialized for overriding such default rules. We extend this model to 'semantically rich domains' by performing a divided-visual-field experiment on 16 chessmasters. Such subjects are able to recall and recognize complex chess positions by chunking the basic elements of the stimuli--the chess pieces--into meaningful groupings according to certain rules that are specific to the semantic structure of the chess domain. We show that the right hemisphere is superior to the left at parsing according to the default rules of chess chunking, but that the left hemisphere is superior to the right at grouping pieces together in violation of those rules. These results suggest that the right hemisphere is better able to acquire and apply new sets of default parsing rules for specific contexts. We conclude, consistent with other neuropsychological evidence, that the right hemisphere is critical for chess skill.

Two types of image generation: evidence for left and right hemisphere processes.

The results from seven experiments provide evidence that visual mental images can be generated by either the left or right cerebral hemisphere, but in different ways. Subjects were cued to form images within a grid or within a set of four corner brackets; a single X mark was enclosed within each stimulus, and the subjects were to determine whether the X mark was enclosed within each stimulus, and the subjects were to determine whether the X mark would have fallen on an imaged pattern. When subjects memorized descriptions of how parts were arranged, they could later form images of the composite pattern when cued in the right visual field (left hemisphere) more accurately than when they were cued in the left visual field (right hemisphere). In contrast, when subjects memorized individual segments on a screen, and 'mentally glued' them into a single pattern, they later could form images more accurately, at least in some circumstances, when cued in the left visual field. These results were predicted by the theory that images are built up by arranging parts, and that two different processes can be used to arrange them. One process uses stored descriptions to arrange parts, and is more effective in the left cerebral hemisphere; the other process uses stored memories of metric positions to arrange parts, and is more effective in the right cerebral hemisphere. Convergent evidence was obtained by having subjects memorize letters in grids (which are easily encoded using descriptions of the positions of segments) or within a space delineated by four brackets (which require memorizing the precise positions of the segments). Subjects were relatively more accurate when cued in the left visual field with bracket stimuli, but tended to be relatively more accurate when cued in the right visual field with grids stimuli. Control experiments showed that this finding was not due to hemispheric differences in the ease of forming images at different sizes or differences in the ease of perceptually encoding the probes.

Effects of moderate physical training on prednisone-induced protein wasting: a study of whole-body and bone protein metabolism.

This study investigated the possibility of preventing prednisone-induced protein wasting by regular physical activity. Eight healthy untrained volunteers took prednisone (30 mg/d for nine days), once after a 4-week exercise program that consisted of jogging 2.5 miles four times a week, and once without exercise. Whole body protein turnover was measured from the 15N enrichment plateau of urinary ammonia during ingestion of 15N glycine at hourly intervals. Whole-body protein synthesis and breakdown were derived from nitrogen flux, nitrogen intake, and urinary nitrogen elimination. Muscle myofibrillar protein breakdown was explored by measuring urinary 3-methylhistidine excretion. Bone protein metabolism was studied by measuring serum bone GLA protein (BGP), a specific marker of bone protein synthesis, and urinary elimination of hydroxyproline, an index of bone resorption. Whole-body protein turnover was significantly increased by exercise and prednisone (+19% and +17%, respectively); this effect was related to increased protein synthesis during exercise training (+27%, P less than .01) and to increased protein breakdown during prednisone administration without exercise (+21%, P less than .05). In contrast, values of protein turnover, synthesis, and breakdown were not different from control when the subjects took prednisone after training. Urinary excretion of 3-methylhistidine was decreased (-15%, P less than .05) at the end of the prednisone administration period but was identical to the control value when the subjects took prednisone in association with exercise. In contrast, serum BGP was significantly decreased by prednisone, with or without exercise (-35%, P less than .001). These data suggest that moderate exercise training can prevent, at least in part, the protein loss induced by prednisone.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors.

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.

Muscarinic-induced modulation of potassium conductances is unchanged in mouse hippocampal pyramidal cells that lack functional M1 receptors.

Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) increases excitability of pyramidal cells by inhibiting several K+ conductances, including the after-hyperpolarization current (Iahp), the M-current (Im), and a leak K+ conductance (Ileak). Based on pharmacological evidence and the abundant localization of M1 receptors in pyramidal cells, it has been assumed that the M1 receptor is responsible for mediating these effects. However, given the poor selectivity of the pharmacological agents used to characterize these mAChR responses, rigorous characterization of the receptor subtypes that mediate these actions has not been possible. Surprisingly, patch clamp recording from CA1 pyramidal cells in M1 knockout mice revealed no significant difference in the degree of inhibition of Iahp, Im, or Ileak by the mAChR agonist, carbachol (CCh), as compared with wildtype controls. In addition, the M1-toxin was not able to block CCh's inhibition of the Iahp, Im, or Ileak These data demonstrate that the M1 receptor is not involved in increasing CA1 pyramidal cell excitability by mediating ACh effects on these K+ conductances.

Role of the M1 receptor in regulating circadian rhythms.

Cholinergic stimuli are potent regulators of the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Using a brain slice model, we have found that the SCN clock is subject to muscarinic regulation, a sensitivity expressed only during the night of the clock's 24-h cycle. Pharmacological and signal transduction characteristics are compatible with a response mediated by an M1-like receptor. Molecular manipulation of muscarinic receptors will provide important insights as to the receptor subtype(s) regulating circadian rhythms.

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.

Alteration of cardiovascular and neuronal function in M1 knockout mice.

We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.

Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice.

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Elucidating the role of muscarinic receptors in psychosis.

Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.