John D. McLaren

Characterization of implantable microfabricated fluid delivery devices

The formal characterization of the performance of microfluidic delivery devices is crucial for reliable in vivo application. A comprehensive laboratory technique was developed and used to optimize, calibrate and validate microfabricated fluid delivery devices. In vivo experiments were carried out to verify the accuracy and reliability of the pressure driven devices. Acute guinea pig experiments were conducted to measure the response to /spl alpha/-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid, an excitatory neurotransmitter, at multiple locations in the inferior colliculus. A nondimensional parameter, Q/spl tilde/, was successfully used to classify devices in terms of geometry alone (i.e., independent of fluid properties). Functional devices exhibited long-term linearity and reliability in delivering single phase, Newtonian fluids, in discrete volumes with a resolution of 500 picoliters at less than 0.45 lbf/in/sup 2/ (30 mbar) pressure drop. Results for non-Newtonian fluids are not presented here. The acute results showed a proportional increase in the firing rate for delivered volumes of 2 nL up to 10 nL (at rates of between 0.1 and 1 nL/s). Flow characteristics are maintained during acute experiments and post-implant. A control experiment conducted with Ringer solution produced negligible effects, suggesting the results to be truly pharmacological. The experimental techniques employed have proven to be reliable and will be used for future calibration and testing of next generation chronic microfluidic delivery devices.

FORMATION OF A TOXIC METABOLITE FROM GENTAMICIN BY A HEPATIC CYTOSOLIC FRACTION

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.

The effect of post type and length on the fracture resistance of endodontically treated teeth

STATEMENT OF PROBLEMnFew studies have been conducted to determine a correlation between the flexural modulus of metal and fiber-reinforced posts and the fracture resistance and failure mode of teeth restored with posts. Questions remain as to whether a longer post length or a post with a higher flexural modulus will significantly improve the fracture resistance of a tooth restored with a prefabricated post and core.nnnPURPOSEnThe purpose of this study was to compare the fracture resistance and mode of failure of endodontically treated teeth restored with 3 different post systems, including 2 fiber-reinforced posts (Light-Post and Snowlight) and a stainless steel post (ParaPost XP).nnnMATERIAL AND METHODSnSeventy single-rooted premolars were sectioned at the cemento-enamel junction and then endodontically treated. Teeth were distributed into 7 groups. Three different prefabricated posts were cemented into a post space either 5 or 10 mm in depth, and composite resin (ParaPost ParaCore automix) cores were fabricated. A composite resin core group without a post served as a negative control. Specimens were loaded at 90 degrees to the longitudinal axis until ultimate failure occurred. An initial failure load and mode of failure were also recorded. Statistical analysis was performed for initial and ultimate failure loads of groups by using 2-way ANOVA (P=.05).nnnRESULTSnThe groups with ParaPost XP posts demonstrated significantly higher initial and ultimate mean failure loads when compared with the fiber-reinforced post groups. The highest mean (SD) initial failure load was with the ParaPost XP group with a 10-mm post length (170.05 (60.08) N), and the lowest was with the Snowlight group with the 5-mm post length (62.85 (18.47) N).nnnCONCLUSIONSnThe stiffness and the load to initial fracture of the teeth restored with ParaPost XP posts were higher compared with the fiber-reinforced post groups.

In vivo release of neuroactive amino acids from the inferior colliculus of the guinea pig using brain microdialysis

Microdialysis techniques were used to measure in vivo release of neuroactive amino acids from the central nucleus of the inferior colliculus (ICC) in anesthetized guinea pigs. Concentric dialysis probes were implanted in the ICC and perfused with Ringer solution of various compositions at a flow rate of 2.0 microliters/min. Consecutive 10-min fractions of the dialysate were collected for up to 3 h under different experimental conditions, frozen and assayed for amino acid content by high performance liquid chromatography (HPLC). There was an initial high outflow of amino acids which declined to stable baseline levels after 2 h. Following this stabilization period, perfusion with a medium containing 100 mM KCl produced an increase in the extracellular levels of aspartate (Asp), glutamate (Glu), gamma-aminobutyric acid (GABA) and glycine (Gly). Only the increases in GABA and Gly were statistically significant. None of the increases occurred in the presence of 2.0 mM cobalt suggesting the release of amino acids is calcium dependent. Histological examination revealed that tissue damage was minimal and largely confined to the immediate vicinity of the probes. We were also able to show that the blood brain barrier (BBB) appeared to heal 2 h after probe implantation. Thus, following intravenous injection of [3H]alpha-aminoisobutyric acid (AIB), which does not cross the intact BBB, no isotope was recovered in the dialysate. These results demonstrate that microdialysis is a unique and suitable method to monitor changes in the extracellular levels of amino acid neurotransmitters in a central auditory structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5′-triphosphate synthetase

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5′-triphosphate (CTP) and deoxycytidine 5′-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5′-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5′-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5′-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.

Potassium-induced release of endogenous glutamate and two as yet unidentified substances from the lateral line of Xenopus laevis.

The release of endogenous glutamate and other primary amines from the lateral-line of Xenopus laevis was studied using an in vitro superfusion technique and high performance liquid chromatography. Potassium stimulation (50 mM KCl) applied to 60 or 120 lateral-line organs dissected from the skin and pooled in a perfusion chamber induced the release of glutamate and aspartate. The release of aspartate was smaller than that of glutamate and more variable. A variable release of two, as yet, unidentified substances was also detected. In low calcium (0.1 mM CaCl2), high magnesium (10 mM MgCl2) solution, 50 mM potassium failed to induce an increase in glutamate, aspartate and the two unknowns, suggesting they are released in a transmitter-like manner. The technique presents a new and simple method for studying transmitters in hair-cell systems. Although other interpretations are possible, the results are consistent with the hypothesis that glutamate is a hair-cell transmitter and suggest a potential role for other substances in the transduction process, perhaps as neuromodulators.

Effect of aging on myo-inositol and phosphoinositide metabolism in the cochlear and vestibular sensory epithelia of the rat

Neurotransmission and transmembrane signaling are among the cellular mechanisms affected in the aging nervous system. In the inner ear, the phosphoinositide second messenger cascade is of particular interest as a target of the aging process. In both the cochlear (CSE) and vestibular sensory epithelia (VSE), the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to the second messenger inositol 1,4,5-trisphosphate (InsP3) is coupled to muscarinic cholinergic and P2y purinergic receptors and may be linked to calcium homeostasis. The present study compared the turnover of phosphoinositides (PtdInsPs), receptor-mediated release of inositol phosphates (InsPs), and concentrations of endogenous myo-inositol in the CSE and VSE of young (3 months) and aged (24 months) Fischer-344 rats. In the aged rat, there was a significant increase in [3H]inositol incorporation (per mass of protein) into PtdInsPs plus InsPs in both sensory epithelia while the protein content remained unchanged. In contrast, no age-dependent differences were found when pre-labeled [3H]PtdInsPs were chased with non-radiolabeled myo-inositol indicating that the turnover of these lipids was unaffected. The cholinergic receptor agonist carbamylcholine and the P2 purinergic receptor agonist adenosine 5-O-(3-thiotriphosphate) stimulated the release of [3H]InsPs two- to six-fold in both organs. This agonist-stimulated release of [3H]InsPs (per mass of protein) was significantly higher in aged animals. However, when the same stimulation was expressed as per cent of control values, there was no age-dependent difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts

SummaryThe synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP.The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used.De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition.A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.

The kinetics and feedback inhibition of cytidine 5'-triphosphate synthetase in wild-type and mutant Chinese hamster cells.

The kinetics and cytidine 5′-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5′-triphosphate (UTP). Three of the mutants had Km app and S50 valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.