Active microelectronic neurosensor arrays for implantable brain communication interfaces.

We have built a wireless implantable microelectronic device for transmitting cortical signals transcutaneously. The device is aimed at interfacing a cortical microelectrode array to an external computer for neural control applications. Our implantable microsystem enables 16-channel broadband neural recording in a nonhuman primate brain by converting these signals to a digital stream of infrared light pulses for transmission through the skin. The implantable unit employs a flexible polymer substrate onto which we have integrated ultra-low power amplification with analog multiplexing, an analog-to-digital converter, a low power digital controller chip, and infrared telemetry. The scalable 16-channel microsystem can employ any of several modalities of power supply, including radio frequency by induction, or infrared light via photovoltaic conversion. As of the time of this report, the implant has been tested as a subchronic unit in nonhuman primates ( approximately 1 month), yielding robust spike and broadband neural data on all available channels.

Development of an integrated microelectrode/microelectronic device for brain implantable neuroengineering applications.

An ultra-low power analog CMOS chip and a silicon based microelectrode array have been fully integrated to a microminiaturized "neuroport" for brain implantable neuroengineering applications. The CMOS IC included preamplifier and multiplexing circuitry, and a hybrid flip-chip bonding technique was developed to fabricate a functional , encapsulated microminiaturized neuroprobe device. As a proof-of-concept demonstration, we have measured local field potentials from thalamocortical brain slices of rats, suggesting that the new neuroport can form a prime platform for the development of a microminiaturized neural interface to the brain in a single implantable unit.

Photovoltaic energy converter as a chipscale high efficiency power source for implanted active microelectronic devices.

We report the development of a microscale photovoltaic energy converter which has been designed and implemented to deliver power to CMOS-based microelectronic chips. The design targets the delivery of voltages on the order of 3V with power levels in excess of 10 mW. The geometry of the prototype device, which has been fabricated and tested, is specifically designed for coupling to an optical fiber, to facilitate remote power delivery in implantable component environment.

Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

A comparison of human and killer whale platelet fatty acid composition.

Activation of blood platelets and their subsequent aggregation results from the interactions of several complex metabolic pathways. Considered to be of critical importance are the platelet lipids. Subsequent to platelet activation, several membrane lipids undergo hydrolysis and the free fatty acids are metabolized to prostanoids which mediate platelet function in response to vascular injury. It is conceivable then, that differences in platelet membrane fatty acid content could result in significant differences in platelet responses to aggregatory stimuli, especially between species. The objective of this study was to identify specific differences in fatty acid content between human and killer whale platelets. Blood was collected, washed platelets were prepared, and platelet fatty acids were extracted. Methyl esters of the extracted fatty acids were analyzed by gas chromatography and reported as relative concentrations. Analysis of the data revealed significant differences between the two species for several relevant fatty acids, i.e. 16:0 (P < 0.05), and 18:0, 18:1, 18:2, and 20:4 (P < 0.001). The differences in platelet fatty acid composition and concentration may explain at least some of the differences in platelet function which have previously been identified between these species.

Hyponatremia with venlafaxine.

OBJECTIVE: To describe a patient with hyponatremia associated with venlafaxine therapy. CASE SUMMARY: A 92-year old white woman who was receiving venlafaxine for management of depression was found to have hyponatremia. A detailed workup confirmed the diagnosis of syndrome of inappropriate antidiuretic hormone secretion (SIADH). A temporal relationship between initiation of venlafaxine therapy and the onset of hyponatremia indicated it as the probable cause. Venlafaxine was discontinued, and hyponatremia resolved with a few weeks. DISCUSSION: Hyponatremia has been reported with selective serotonin-reuptake inhibitors (SSRIs). Serotonin has been reported to elevate concentrations of vasopressin in animal models. Venlafaxine is a potent inhibitor of serotonin reuptake and may have adverse effects similar to those of SSRIs. CONCLUSIONS: We report a case of hyponatremia probably caused by venlafaxine. Awareness of this potential problem would be helpful to clinicians and should be considered in the differential diagnosis of hyponatremia.

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

Crystal structure of recombinant pea cytosolic ascorbate peroxidase.

The crystal structure of recombinant pea cytosolic ascorbate peroxidase has been refined to an R = 0.19 for data between 8.0 and 2.2 A resolution and magnitude of F > or = 2 sigma(magnitude of F). The refined model consists of four ascorbate peroxidase monomers consisting of 249 residues per monomer assembled into two homodimers, with one heme group per monomer. The ascorbate peroxidase model confirms that the pea cytosolic enzyme is a noncovalent homodimer held together by a series of ionic interactions arranged around the 2-fold noncrystallographic dimer axis. As expected from the high level of sequence identity (33%), the overall fold of the ascorbate peroxidase monomer closely resembles that of cytochrome c peroxidase. The average root mean square differences for 137 helical alpha-carbon atoms between the four ascorbate peroxidase monomers and cytochrome c peroxidase and for 249 topologically equivalent alpha-carbon atoms are 0.9 and 1.3 A, respectively. The active site structures are also the same, including the hydrogen-bonding interactions between the proximal His ligand, a buried Asp residue, and a Trp residue, whose indole ring is parallel to and in contact with the proximal His ligand just under the heme ring. This proximal Trp residue is thought to be the site of free radical formation in cytochrome c peroxidase compound I and is also essential for enzyme activity. The corresponding Trp in ascorbate peroxidase, Trp179, occupies exactly the same position. The most interesting, and possibly functionally important, difference between the two peroxidases is the presence of a cation binding site in ascorbate peroxidase located approximately 8 A from the alpha-carbon atom of Trp179.

Identification of a porphyrin pi cation radical in ascorbate peroxidase compound I.

Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the ascorbate peroxidase Fe3+ resting state and to compare the reaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase. Because ascorbate peroxidase has a Trp residue in the proximal heme pocket at the same location as the Trp191 compound I free radical in cytochrome c peroxidase [Patterson, W. R., & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated that ascorbate peroxidase compound I might also contain a Trp-centered radical. However, the ascorbate peroxidase compound I EPR spectrum is totally different from that of cytochrome c peroxidase. Immediately after the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidase compound I exhibits an axial resonance extending from g perpendicular = 3.27 to g parallel approximately 2 that disappears within 30 s, presumably due to endogenous reduction of compound I. In contrast, cytochrome c peroxidase compound I exhibits a long-lived g approximately 2 signal associated with the Trp191 cation free-radical [Houseman, A. L. P., et al. (1993) Biochemistry 32, 4430-4443]. Recently, the 2 K EPR spectrum of a catalase compound I was found to exhibit a broad signal extending from g perpendicular = 3.45 to g parallel approximately 2 and was interpreted as a porphyrin pi cation radical [Benecky, M. J., et al. (1993) Biochemistry 32, 11929-11933]. On the basis of these comparisons, we conclude that ascorbate peroxidase forms an unstable compound I porphyrin pi cation radical, even though it has a Trp residue positioned precisely where the Trp191 radical is located in cytochrome c peroxidase.

Characterization and crystallization of recombinant pea cytosolic ascorbate peroxidase.

An Escherichia coli expression system has been developed for pea cytosolic ascorbate peroxidase (APX). The enzyme was expressed as a fusion product with the E. coli maltose-binding protein for rapid, affinity chromatography purification. Recombinant ascorbate peroxidase (rAPX) was purified by tryptic digestion to separate the maltose-binding protein from rAPX followed by three chromatographic steps. The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots. Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center. rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected.

Aggregation of killer whale platelets.

The aggregation of blood platelets is a crucial step in normal hemostasis for all mammals. Circulating platelets are sensitive to a large variety of physiologic and non-physiologic stimulants, some of which are formed or exposed in conjunction with vascular damage or endothelial cell denudation. In addition, drastic pressure changes activate human platelets. Killer whale platelet function, on the other hand, is very intriguing since these animals do not seem to experience untoward platelet reactions during or after diving to great depths, nor do they experience abnormal bleeding associated with sub optimal platelet function. We examined this concept and determined that killer whale platelets, in response to ADP, PAF, and arachidonic acid, appeared to aggregate normally during the first 2-5 minutes after addition of the agonist, but had completely disaggregated at 10 minutes. Collagen- and A23187-induced aggregation appeared normal and complete within 10 minutes, while there was no response to epinephrine or ristocetin. Thromboxane production by killer whale platelets appears to be quantitatively similar to that produced by human platelets in response to ADP and PAF and exceeded that produced by human platelets when collagen was used as the agonist. In summary, this study reports a reduced platelet aggregation reaction in killer whales in response to several platelet agonists which does not appear to be related to the generation of thromboxane. This phenomenon may serve a protective role in these mammals by preventing thrombosis during diving and resurfacing.

Acute hemodynamic and respiratory effects of amniotic fluid embolism in the pregnant goat model.

OBJECTIVE: Our purpose was to determine the acute-phase central hemodynamic and respiratory effects of raw, filtered, filtered and boiled, and meconium-containing amniotic fluid. STUDY DESIGN: Pregnant goats (Capra hircus) in the last one third of pregnancy were given freshly collected autologous amniotic fluid in a volume of 2.5 ml/kg of body weight. Observations were then made at 10, 30, 60, 120, and 180 minutes after amniotic fluid embolism. Pulmonary artery catheters and femoral artery lung water catheters were placed for specimen and data collection. RESULTS: Marked pressor responses were observed in both the pulmonary and systemic circulations with all amniotic fluid infusions. The pressor response was similar with raw, filtered, and filtered and boiled amniotic fluid. The pressor response seen with amniotic fluid containing meconium was significantly greater than that seen with the other forms. No significant effects were observed on cardiac or respiratory function except in the meconium group, where transient left ventricular dysfunction was accompanied by an acute increase in extravascular lung water and dysoxia. CONCLUSIONS: The Capra hircus model is appropriate for the further study of amniotic fluid embolism. The acute pressor effects are transient and involve both the systemic and pulmonary circulations. Left ventricular dysfunction and dysoxia were observed only with embolism of amniotic fluid containing meconium.

Creatine kinase and creatine kinase isoenzymes as a marker of uterine activity.

Creatine kinase (CK) and CK isoenzymes are known to fluctuate in labor. Reliable information about the longitudinal changes of CK and CK isoenzymes during labor is sparse. Nevertheless, they have been used to direct care in women with cardiopulmonary disease and preterm labor requiring tocolysis. This study evaluated fluctuations of CK and its isoenzymes longitudinally across labor in 49 women. Blood samples were obtained at 33 to 34 weeks' estimated gestational age, on admission in labor at 3 cm or less dilation, 8 cm to complete dilation, and postpartum in the recovery room. Specimens were analyzed for total CK, CK-MM, CK-MB, and CK-BB activity. CK levels increased for all peripartum patients (p < 0.001). CK activity at 3 cm was greater than at 34 weeks (p < 0.01). Furthermore, the early rise in CK activity was greater in those in active labor compared with those who required oxytocin stimulation (p < 0.001). CK values at 8 cm and postdelivery (mean IU/liter) were often above nonpregnant norms. The early rise of CK in spontaneously laboring patients versus those requiring oxytocin augmentation may represent a difference in uterine activity. Nonpregnant normative data for CK is not appropriate when assessing cardiovascular side effects of betamimetic therapy.

New extraction procedure and high-performance liquid chromatographic method for analyzing polyethylene glycol-400 in urine.

We describe a new, highly efficient method for extracting polyethylene glycol-400 from urine and for its analysis by isocratic reversed-phase high-performance liquid chromatography. This method is an improvement over previously published methods in that it does not require the use of ion-exchange resins and lyophilization prior to extraction, nor does it require the separation and analysis of the individual polymers of polyethylene glycol. The procedure described in this report entails extraction with a salt-solvent combination of ammonium sulfate and dichloromethane and analysis by reversed-phase high-performance liquid chromatography. The lower limit of detection was approximately 0.25 g/l with a 2-ml urine sample. Analytical recoveries of polyethylene glycol-400 added to urine at 2.5 and 5.0 g/l averaged 97 and 96%, respectively (n = 10). Within- and between-day coefficients of variation were less than 5% at 2.5 and 5.0 g/l. Studies of various urine samples from patients receiving polyethylene glycol-400 revealed no interferences from other urine substances.

A comparison of human and porcine platelet fatty acid composition.

1. Human and pig platelets were analyzed for lipid composition by gas chromatography. 2. In comparing the two species' platelets, we noted significant differences in several lipid concentrations, some of which involved major lipid constituents. 3. The lipids for which there are significant differences in composition may contribute to those variations in platelet responses to stimulation demonstrated to exist between species.

A comparison of human and canine platelet fatty acid composition.

1. Human and canine platelets were analysed for lipid composition by gas chromatography. 2. In comparing the two species' platelets, there were several lipids for which there were significant differences in lipid concentration. 3. The results indicate that human and canine platelets are quite similar in platelet lipid composition; however, the lipids for which there are significant differences may contribute to variations between species in platelet responses to stimulation.

Levels of leakage radiation from electron collimators of a linear accelerator.

The leakage radiation from electron applicators used with our linear accelerator has been measured. For the applicators 6 X 6 to 25 X 25 cm size, the leakage was measured in the plane of the patient and on the sides of the applicators with the available electron energies of 6, 9, 12, 15 and 18 MeV. The levels were significant. The highest leakage on the side was for the combination of 6 X 6-cm applicator and 9-MeV electrons (32%) and in the plane of the patient for 25 X 25-cm applicator with 18 MeV (10%) relative to the peak dose. Adding lead 1-2 mm, at appropriate locations inside the applicators has reduced the leakages to acceptable levels without affecting the beam parameters.

Absent platelet aggregation with normal fibrinogen binding in basset hound hereditary thrombopathy.

Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) initially displayed a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa), and therefore are more accurately described as thrombopathic. The presence of normal quantities of GPIIb-IIIa, however, did not rule out the possibility of a functionally abnormal glycoprotein complex which would be unable to bind radio-labeled fibrinogen. Therefore, fibrinogen binding in BHT platelets was evaluated. Platelets from BHT and normal dogs were activated with 1 x 10(-5) M ADP in the presence of 125I-fibrinogen and the surface-bound radioactivity was quantitated. The amount of fibrinogen bound by BHT dog platelets was not significantly different than that bound by normal dog platelets. Platelets from dogs with BHT bound 30,282 +/- 3,133 and normal dog platelets bound 31,664 +/- 2,772 molecules of fibrinogen per platelet. The quantitatively normal GPIIb-IIIa complex binds fibrinogen in normal amounts and does not seem to represent the abnormality responsible for the aggregation defect in BHT platelets. Therefore, other factors central to normal platelet function and related to platelet aggregation must be considered.

High-performance liquid chromatographic method for the simultaneous determination of the enantiomers of carvedilol and its O-desmethyl metabolite in human plasma after chiral derivatization.

Quantitative methodology for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of carvedilol, a new multiple-action antihypertensive agent exhibiting both vasodilator and beta-blocking activity, and its active metabolite, O-desmethylcarvedilol, in human plasma is described. The method involves reversed-phase solid-phase extraction of the analytes, followed by derivatization of the extract with the chiral reagent, 2,3,4,6,-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate and injection of the resultant diastereoisomers onto a reversed-phase HPLC column coupled to a fluorescence detector. Both pairs of diastereoisomers formed are completely resolved within 12 min (resolution for the respective pairs is 2.26 and 3.32) and the baseline is clean and free from extraneous peaks. The assay is linear over the range 0.6-80 ng/ml of human plasma with a lower limit of detection of approximately 100 pg on-column for each of the enantiomers. The method can be adapted for a number of structural analogues of carvedilol and is currently applied in support of preclinical and clinical studies of the drug.