A single-channel implantable microstimulator for functional neuromuscular stimulation

This paper describes a single-channel implantable microstimulator for functional neuromuscular stimulation. This device measures 2 x 2 x 10 mm3 and can be inserted into paralyzed muscle groups by expulsion from a hypodermic needle. Power and data to the device are supplied from outside by RF telemetry using an amplitude-modulated 2-MHz RF carrier generated using a high-efficiency class-E transmitter. The transmitted signal carries a 5-b address which selects one of the 32 possible microstimulators. The selected device then delivers up to 2 microC of charge store in a tantalum chip capacitor for up to 200 microseconds (10 mA) into loads of < 800 omega through a high-current thin-film iridium-oxide (IrOx) electrode (approximately 0.3 mm2 in area). A bi-CMOS receiver circuitry is used to: generate two regulated voltage supplies (4.5 and 9 V), recover a 2-MHz clock from the carrier, demodulate the address code, and activate the output current delivery circuitry upon the reception of an external command. The overall power dissipation of the receiver circuitry is 45-55 mW. The implant is hermetically packaged using a custom-made glass capsule.     

Quantitation of intracellular [Na+] in vivo by using TmDOTP5- as an NMR shift reagent and extracellular marker

A method is presented to measure the absolute concentration of intracellular Na+ ([Na+]i) in vivo by using interleaved 23Na- and 31P-nuclear magnetic resonance (NMR) spectroscopy and TmDOTP5- as shift reagent and chemical marker of tissue extracellular space (ECS). The technique was used to determine [Na+]i and relative ECS in livers of control rats (21 +/- 3 and 0.11 +/- 0.02 mM, respectively) and in rats exposed to carbon tetrachloride (103 +/- 29 and 0.23 +/- 0.03 mM, respectively). The NMR measurements were confirmed independently on excised tissue samples by using atomic absorption spectroscopy. The results confirm that TmDOTP5- can be used as a combined cation shift reagent and ECS marker, thereby allowing quantitation of [Na+]i in vivo by NMR.     

Effects of endurance training in the leopard shark, Triakis semifasciata

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed.     

A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology

A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.     

Species heterogeneity of Gly-11 gramicidin A incorporated into sodium dodecyl sulfate micelles

Evidence is presented for species heterogeneity of the gly-11 analog of gramicidin A incorporated into sodium dodecyl sulfate (SDS) micelles. The evidence for species heterogeneity has been obtained using one-dimensional (1D) 1H NMR spectroscopy. The 1D spectra of the indole NH moiety of tryptophans 9, 13, and 15 show the presence of more than one species. It has been found that the heterogeneity is dependent upon the gly-11/SDS molar ratio. At high SDS concentration (i.e., gly-11/SDS of 3 mM/700 mM) the heterogeneity almost completely disappears. The temperature dependence of these 1H NMR signals suggests that the two species do not interconvert. The results of nuclear Overhauser effect spectroscopy NMR experiments indicate that one species is embedded within the micelle, while the other is nearer the aqueous interface. The importance of side chain interactions with the membrane environment in producing stable, solubilized species of small peptides in SDS micelles is illustrated.     

Method for treating and processing whole chick embryos for autoradiography, immunocytochemistry and other techniques

A method is presented for in situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions "in ovo." The chick embryo is placed in a "shell-less" culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo+membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

Fatal bleomycin pulmonary toxicity in the west of Scotland 1991-95: a review of patients with germ cell tumours

This paper describes a method for matching high-temperature superconducting nuclear magnetic resonance (NMR) signal pickup coils to field-effect transistor (FET) preamplifiers using amplifier-specific noise figure and gain parameters to obtain a matching compromise that improves gain flatness for a given permissible noise figure. A technique for obtaining these parameters is also described in an appendix.     

FRP-Confined Concrete Model

A uniaxial model for concrete confined with fiber-reinforced polymers (FRP), but also with steel jackets or conventional transverse reinforcement, is presented. The model, which is suitable to be inserted into fiber-type beam-column models, explicitly accounts for the continuous interaction with the confining device due to the lateral strain of concrete, through an incremental-iterative approach. The relation between the axial and lateral strains is implicitly derived through equilibrium between the (dilating) confined concrete and the confining device. This relation allows one to trace the state of strain in the jacket and to detect its failure. The model is compared with a set of experimental tests and shows very good agreement in both the stress-strain and the stress-lateral strain response. Evidence of the main aspects of the behavior of FRP-confined concrete is given that points out differences in the response when using fiberglass or carbonfiber. Predictive equations to determine the ultimate strength and strain of concrete confined with FRP are derived and tested on a number of experimental data.     

The peripherally inserted central catheter: a retrospective look at three years of insertions

The advent of intravenous therapies administered in the home setting has created a demand for the peripherally inserted central catheter (PICC). A variety of PICCs is now on the market, offering choices of insertion methods as well as size and length. This article represents a retrospective look at 858 PICCs inserted over a 3-year period, using three insertion methods: 1) the Seldinger technique, 2) the peel-away sheath, and 3) the break-away needle. A brief history of PICCs along with problems encountered, an account of PICCs in immune-suppressed patients, and principles of catheter management are presented.     

Theoretical studies of an exceptionally stable RNA tetraloop: observation of convergence from an incorrect NMR structure to the correct one using unrestrained molecular dynamics

We report on the results of five independent and unrestrained molecular dynamics simulations of an RNA tetraloop, r(GGACUUCGGUCC), and its related structures with the loop UUCG sugars changed to deoxyribose. Two separate NMR structures have been reported for the loop portion of this molecule, with the second refinement resulting in a slightly different and more accurate conformation for the loop. The root-mean-square deviation (RMSd) between the two NMR structures, for the loop portions only, is 2.5 A. Our simulations, starting from the two NMR structures, demonstrate that this tetraloop is a very stable and rigid structure with both nanosecond length simulations staying very close to the initial structures. Additionally, both simulations preserved most, if not all, of the NMR-derived interactions and violated very few of the nuclear Overhauser effect (NOE)-derived distances used in the structure refinements. However, when the two NMR structures were simulated with deoxyriboses in the loops instead of the native riboses, the flexibility of the systems increased and we observed a conversion from the incorrect to the correct loop conformation in the simulation which started in the incorrect loop conformation. When the riboses were subsequently re-introduced back into the structure which underwent the conversion, the agreement between this simulation and the one starting from the correct NMR structure was a remarkably low 0.5 A, demonstrating an almost complete convergence from the incorrect to the correct structure using unrestrained molecular dynamics.     

Selective long-range DEPT NMR technique and its application to structural study of glycoside]

This paper reports a new NMR technique: selective long-range DEPT. The result obtained showed that the technique can be used to assign NMR signal of 13C, 15N and 31P nuclei, and connect the spin systems separated by quaternary carbon or heteroatom. This paper deals mainly with applying the technique for determining the glycosidation position in aglycone moiety and sequence of sugar moiety in a natural glycoside.     

A compensation method for the disturbance in the temperature field caused by subsurface thermocouples

The presence of a subsurface thermocouple hole can lead to a local disturbance of the temperature field in a sample and to a discrepancy between the thermal history measured by the thermocouple and the thermal history in an area without a thermocouple. This phenomenon also leads to errors in the surface heat flux calculated using an inverse heat conduction model, which relies on the acquired thermal history data. This article analyzes the factors that influence the temperature discrepancy caused by a subsurface thermocouple hole. A technique called the “equivalent depth” technique is proposed to compensate for the disturbance of the temperature field due to the presence of a thermocouple hole. This technique involves off-setting the location of the thermocouple position based on the radius of the thermocouple hole. A verification of this technique for the calculation of the surface heat flux using an inverse heat conduction model with measured thermal history data is presented.     

Structural information on a cecropin-like synthetic peptide, Shiva-3 toxic to the sporogonic development of Plasmodium berghei

This study is a contribution towards the understanding of the mode of action of Shiva-3 and more generally that of cecropin-like peptides. Structural information on Shiva-3 (a cecropin-like synthetic peptide) in water and in a membrane-mimicking environment (trifluoroethanol alcohol, SDS) were obtained using analytical centrifugation, CD and NMR spectroscopies. A total of 20 converged structures were retained on the basis of 197 non-redundant experimental constraints, including 166 distance constraints from the nuclear Overhauser effect measurements and 31 dihedral angle restraints derived from the purged COSY experiments. Some results obtained in presence of SDS are also presented. The toxic effects of the peptides obtained by cleavage (trypsin and lysine-C hydrolysis) of Shiva-3 on Escherichia coli and on Plasmodium berghei sporogonic stages are reported. Biological effects are discussed in relation to the calculated structure. The antiparasite activity and the low mosquito toxicity of Shiva-3 make this peptide a good candidate for genetic transformation of mosquito vectors which warrants further studies aimed at the improvement of the molecule.     

Arthrodesis of finger joints by dynamic external compression using dorsoventral Kirschner wires and rubber bands

A new method is presented for arthrodesis of small joints in the hand. In this method Kirschner wires are inserted dorsoventrally through the phalanges on either side of the joint, and the external ends bent into hooks. Longitudinal compression is achieved by connecting these hooks with rubber bands. This technique gives a solid and painless immobilization in a proper position, and the latter can be corrected easily when necessary. We arthrodesed 46 joints in 39 patients by this method in 1975 and 1976. The fusion time was 4 to 6 weeks, and the method produced a good bony arthrodesis in 42 of the 46 fingers.     

Unsteady Flow in Hydraulic Networks with Polymeric Additional Pipe

This paper presents results of an experimental and theoretical study on the use of a high density polyethylene (HDPE) additional pipe inserted downstream of a pump in a hydraulic network as a surge suppressor. The experiments consistently show a reduction of the oscillations with respect to the case without a HDPE device, while in the case of a single pumping pipeline the oscillations can be amplified for small volumes of the additional pipe. Previously calibrated mechanical parameters are considered in the mathematical models whose results are compared with experimental results. Both linear elastic and Kelvin-Voigt viscoelastic behavior of the pipe material and both one-dimensional (1D) and quasi-2D flow models are taken into account. The numerical results show that the viscoelastic model better describes the phenomenon, but the elastic model adequately estimates the maximum and minimum oscillations. Furthermore, the results of the quasi-2D model are in better agreement with the experimental maximum and minimum oscillations than those of the 1D model, but the differences are less important than in the case of networks without a HDPE device.     

Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyses but is difficult to prove rigorously. We demonstrate a sensitive method for detecting partially folded species based on using proton chemical shifts as local probes of structure. The coincidence of denaturation curves for probes throughout the molecule is a particularly stringent test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and circular dichroism (CD). This truncated version lacks the residues required for dimerization and is monomeric under the conditions used for NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C resonances. The secondary and tertiary structure of lambda 6-85 is very similar to that reported for the crystal structure of the DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196], as judged by analysis of chemical shifts, amide hydrogen exchange, amide-alpha coupling constants, and nuclear Overhauser enhancements. Thermal and urea denaturation studies were conducted using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fraction denatured versus denaturant concentration obtained from these studies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicating that there are no populated intermediates in the folding of lambda 6-85.     

Intraosseous infusion in an emergency situation: a case report

A patient with a pelvic silicone prosthesis is presented. The sonographic and computed tomographic features in such patients can be confusing and incorrectly interpreted unless the radiologist knows that the prosthesis had been inserted as a radioprotective device.     

N-terminal hydrophobic sorting signals of preproteins confer mitochondrial hsp70 independence for import into mitochondria

The requirement of mitochondrial hsp70 (mt-hsp70) for the import of a series of preproteins containing hydrophobic sorting signals into isolated yeast mitochondria was investigated. Here we demonstrate that the presence of such a sorting signal in proximity to the N-terminal matrix-targeting sequence of a preprotein can secure a translocating polypeptide chain in the import channel in a manner that does not require mt-hsp70 activity. Trapping the translocating chain in this fashion leads to efficient processing by the mitochondrial processing peptidase and to complete translocation across the outer mitochondrial membrane into the intermembrane space. These mt-hsp70-independent effects appear to be exerted at the level of the inner membrane through an interaction of the hydrophobic core of the sorting signal with component(s) of the translocase of the inner membrane. Hydrophobic sorting signals of inner membrane proteins inserted into the membrane from the matrix, as well as those of intermembrane space proteins, are capable of causing this mt-hsp70-independent stabilization, demonstrating that this phenomenon is not unique to those preproteins normally sorted to the intermembrane space.