Somatic mosaicism caused by monoallelic reversion of a mutation in T cells of a patient with ADA-SCID and the effects of enzyme replacement therapy on the revertant phenotype.

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion.

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development.

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ~60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 µg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 µg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).

Wavelet and short-time Fourier transform analysis of electromyography for detection of back muscle fatigue.

Measurement of the time-varying characteristics of the frequency content of trunk muscle electromyography is a method to quantify the amount of fatigue endured by workers during industrial tasks, as well as a tool that may guide the training and rehabilitation of healthy and injured workers. Quantification of the change of signal power within specific frequency ranges may shed greater insight into the fatigue process. Sixteen healthy male subjects performed isometric trunk extension at 70% of their maximum voluntary contraction. Surface electromyography from medial and lateral erector spinae, and latissimus dorsi locations were processed using the short-time Fourier transform (STFT) and wavelet transform. Linear regression quantified the time rate of change of median frequency as well as frequency specific STFT filter and wavelet scale measures. The median frequency from the short-time Fourier transform declined by 22 Hz/min from an initial value of 77 Hz on average. The wavelet and STFT filter measures demonstrated this decline to be caused by a reduction in 209-349 Hz signal power in addition to an increase in 7-88 Hz signal power. A significant reduction in median frequency and significant elevation in 13-22 Hz wavelet signal component was detected in about 90% of the cases, indicating their use for detecting and quantifying fatigue.

Design and assembly of an 8 tesla whole-body MR scanner.

PURPOSE: The purpose of this report is to describe the design and construction of an 8 T/80 cm whole-body MRI system operating at 340 MHz. METHOD: The 8 T/80 cm magnet was constructed from 414 km of niobium titanium superconducting wire. The winding of this wire on four aluminum formers resulted in a total inductance of 4,155 H. Gradient subsystems included either a body gradient or a head gradient along with a removable shim insert. The magnet and gradient subsystems were interfaced to two spectrometers. These provided the control of the gradient amplifiers and the two sets of four RF power amplifiers. The latter provide in excess of 8 kW of RF power from 10 to 140 MHz and 10 kW of RF power from 245 to 345 MHz. A dedicated computer-controlled patient table was designed and assembled. The entire system is located in a clinical setting, facilitating patient-based studies. RESULTS: The 8 T/80 cm magnet was energized without complication and achieved persistent operation using 198.9 A of current, thereby storing 81.5 MJ of magnetic energy. Exceptional performance was observed for nearly all components both in isolation and when combined within the complete system. CONCLUSION: An 8 T/80 cm MRI system has been assembled. The magnet subsystem is extremely stable and is characterized by good homogeneity and acceptable boil-off rates.

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.

Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase.

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix [Stivers, J. T., et al. (1999) Biochemistry 38, 952]. A general acid-base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1' of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k(cat) for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for catalysis. This unprotonated group has a small DeltaH of ionization (-0.4 +/- 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k(cat) for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5-10. Hence, the pK(a) of His187 must be outside this pH range if it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG.

Depolarization of membrane potential and the smooth muscle contraction by isothiocyanatobenzyl imidazoline in guinea-pig stomach circular muscle.

Isothiocyanatobenzyl imidazoline (IBI) produces characteristic slowly developing contraction of many smooth muscle preparations including the circular smooth muscle of the guinea-pig stomach. Changes in the membrane potential were recorded intracellularly, and the muscle contraction induced by IBI was investigated. IBI at 100 micromol/l slowly produced a sustained depolarization of the membrane with a maximum change of approximately 15 mV. This depolarization could not be blocked by 1-hyoscyamine, 100 nmol/l. An imidazoline analogue, oxymetazoline at 1 micromol/l, did not change the resting membrane potential as observed after IBI. Significant membrane depolarization after IBI still occurred in Ca2+-free medium. During IBI-induced depolarization, sudden reduction of Na+ to 30 mmol/l in the medium reduced the depolarization slightly. IBI-induced depolarization was additive with that produced by 20 mmol/l K+ in the medium. In the presence of tetraethylammonium chloride or levcromakalim or nifedipine, IBI continued to depolarize the membrane although functional pharmacological experiments showed that the contractile effects of IBI were significantly inhibited by 30 micromol/l levcromakalim and abolished by 100 nmol/l nifedipine. At 100 micromol/l phentolamine (reported by others as an inhibitor of ATP-sensitive potassium channels) completely blocked IBI-induced contraction. Phentolamine (30 micromol/l) blocked the contractile effects of IBI by 50%. On the other hand, S(-)-Bay K 8644, a voltage-dependent calcium channel activator, was additive with the contractile response of IBI. These results indicated that IBI produced membrane depolarization and contraction of the guinea-pig stomach circular muscle, by a mechanism not involving muscarinic receptors or alpha-adrenoceptors. Even though levcromakalim, an ATP-sensitive potassium channel opener, could not inhibit IBI-induced depolarization, the ATP-sensitive potassium channel and the voltage-dependent calcium channel may be intrinsically linked with the action of IBI.

Wavelet analysis of electromyography for back muscle fatigue detection during isokinetic constant-torque exertions.

STUDY DESIGN: An investigation of the effects of human trunk extensor muscle fatigue on the temporal change in frequency content of the electromyogram as quantified using the Fourier and wavelet transforms during the performance of repetitive dynamic trunk extension. OBJECTIVE: To evaluate whether alterations in the Fourier and wavelet transform measures were consistent with a shift of the signal power to lower frequencies, and to determine which measures were more highly correlated with the decline in maximal trunk extension torque. SUMMARY OF BACKGROUND DATA: Objective assessment of trunk muscle fatigue is likely to play a more important role in the rehabilitation and prevention of low back injuries, given the association between lack of trunk muscle endurance and acquisition of low back pain. Validation of new methods designed to quantify the level of fatigue using the surface electromyogram is necessary before these techniques can be used in industrial rehabilitation settings. The wavelet transform is a recent development in the signal processing of electromyograms that shows promise as a method for assessment of fatigue. METHODS: Trunk muscle electromyograms obtained from study participants performing repetitive isokinetic trunk extension endurance tests were analyzed using the wavelet and the traditional Fourier methods. Trunk extension torque was controlled at 35% and 70% of the participants' maximal voluntary contraction while they exerted at 5 and 10 repetitions per minute. The decline in maximal trunk extension torque was measured once per minute. Linear regression quantified the rate of change in Fourier and wavelet measures caused by fatigue, whereas Pearson's correlation coefficient determined their association with the decline in maximum torque. RESULTS: Changes in the characteristics of the electromyogram were consistent with a shift to lower frequencies: The signal power at higher frequencies was reduced, whereas the power at lower frequencies was elevated. The amount of change was dependent on the task conditions (exertion level and repetition rate). The wavelet-based measures demonstrated as strong an association with the decline in maximal torque output as the Fourier-based measures. CONCLUSIONS: This study demonstrates that assessment of trunk muscle fatigue during isokinetic movementis possible using both Fourier and wavelet measurements. However, the methods were not as likely to change significantly during lower rates of exertion. These methods, when implemented in a controlled setting, may be used to document the rehabilitation process and guide preventive exercise training.

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.

Simulation of cardiac conduction system in distributed computer environment.

A high resolution, three dimensional, computer model of the cardiac conduction system has been developed. Cardiac geometry was constructed from sectional images of VHP project of the National Library of Medicine. The heart was modeled as a matrix of cells that fill its anatomical structure. The intracellular distance was 1 mm and the total number of cells were 457,482. Electrophysiological parameters like action potential, absolute refractory period and conduction velocity were assigned to each of the cells. The pattern of the excitation sequence propagation as well as potentials on the body surface points were computed on a single processor. The working memory and the time for computation of the algorithms were minimized using efficient data structures. The time to compute an excitation sequence over one cardiac cycle was 4 hours. The algorithms were also implemented on a distributed network of personal computers running on a QNX operating system. The speed of computation of the excitation sequence algorithm was improved by a factor of 2.52 when the algorithm was implemented on a network of three Intel-66 MHz machines.

Wavelet analysis of electromyography for back muscle fatigue detection during dynamic constant-torque exertions.

The fatigue of the back muscles appears to be strongly implicated as a risk factor for acquisition of low back pain, which is one of the leading ills of our industrial society. Previously, researchers have successfully measured the level of muscular fatigue by using the Fourier transform to analyze the frequency content of the electromyogram (EMG). However, due to the requirement that the EMG signal be stationary, the Fourier transform is suitable only for the analysis of static muscle exertions in which the muscle is held at constant length and tension. Because the majority of industrial work tasks are not static in nature, new methods for quantifying fatigue during dynamic work are needed. The wavelet transform is a novel, although mathematically well developed, technique for analyzing non-stationary signals that has only recently been applied to the study of EMG. Consequently, the main objective of this project is to develop techniques, using the wavelet transform, for the quantification of back muscle fatigue during dynamic repetitive working conditions.

Wavelet analysis of SAECG to identify patients with conduction defects at risk for sudden cardiac death.

The aim of this study was to determine how Wavelet transform analysis of signal-averaged ECGs can identify patients with conduction defects who are at high risk for development of ventricular tachycardia. In this study, 34 SA-ECGs and programmed electrical stimulation (PES) reports were obtained from the OSU Department of Cardiology Database (1988-1996) and divided into two groups: 17 patients that had inducible monomorphic VT by PES (VT+) and 17 that showed no arrhythmias (VT-). We used Morlet's wavelet to analyze the X, Y, Z, and RMS vector magnitudes in each group. The mean duration from the peak of the RMS vector magnitude to the QRS offset was statistically different with a T value (2-tailed distribution, unequal variance) of 0.033. We noted statistically significant (p < 0.0001) differences in Wavelet energies for 44 msec after the peak of the RMS vector magnitude largest in the Z lead, the first 22 msec, and frequency bins less than 131 Hz. Although no clinical marker could be determined using Wavelet analysis to distinguish the the VT+ from the VT- group, the results from this study show that their SA-ECGs are indeed different even though the optimal analysis has not yet been devised.

Menthol and nonmenthol cigarettes and smoke exposure in black and white women.

Purposes of this investigation were to compare smoke constituent exposure (CO and nicotine boosts) and smoking topography parameters between black and white women, and between women regularly using menthol or nonmenthol cigarettes. A two-factor factorial design with a sample of 37 women stratified by race and menthol or nonmenthol cigarette use was implemented. There were significant main and interaction effects of race and menthol/nonmenthol use on CO boost. Black women had a mean CO boost of 10.1 ppm vs. 7.2 ppm for white women, while women using nonmenthol cigarettes had a higher CO boost (mean = 10.6 ppm) compared to those regularly using menthol cigarettes mean = 6.5 ppm). White menthol smokers had the lowest CO boost of all subgroups. There was a trend for black women to have higher nicotine boost than white women (21.4 ng/ml vs. 15.9 ng/ml). Black women had nonsignificantly higher puff volumes compared to white women (mean = 48.4 vs. 43.5 ml), while nonmenthol smokers had nonsignificantly higher puff volumes than menthol smokers (mean = 48.5 vs. 42.7 ml). Lower CO boost with mentholated cigarettes suggests factors beyond mentholation may affect elevated smoke constituent exposure among black women.

Jaw1, A lymphoid-restricted membrane protein localized to the endoplasmic reticulum.

Jaw1 is a novel lymphoid-restricted gene that is expressed in a developmentally regulated fashion in both the B and T cell lineages. Jaw1 mRNA is abundantly expressed in pre-B and B cell lines with minimal or undetectable expression in plasma cell lines. Pre-T cell lines and normal mouse thymocytes express high levels of Jaw1 mRNA, whereas most mature T cell lines express low levels. Comparison of the mouse and human genes reveals that Jaw1 encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a COOH-terminal transmembrane domain. Jaw1 was localized to the endoplasmic reticulum (ER) of lymphocytes by indirect immunofluorescence and confocal microscopy. When overexpressed in HeLa cells, Jaw1 protein targeted to the ER. In vitro translation of Jaw1 in the presence of canine microsomes demonstrated that Jaw1 is an integral membrane protein of the ER and is oriented on the ER membrane facing the cytosol. Jaw1 is a member of a class of proteins with COOH-terminal hydrophobic membrane anchors and is structurally similar to proteins involved in vesicle targeting and fusion. These findings suggest that the function and/or the structure of the ER in lymphocytes may be modified by lymphoid-restricted resident ER proteins.

Pattern generator system as a versatile visual stimulator.

We have designed and implemented a Motorola 68000 microprocessor-based pattern generator system (PGS) that uses a color video display terminal (VDT) to provide light stimuli to the intact vertebrate retina. This communication is intended for those who are considering acquisition of a commercial retinal stimulator or those who are custom designing their own pattern generator system. The discussion surveys the features to be included as well as design factors which must be considered in such a device. The memory organization of the PGS allows as stimuli multiple, complex patterns consisting of one or more disks, annuli, bars or gratings to flash or modulate in intensity according to a pre-defined function. In addition, patterns can move smoothly in any direction at selectable, uniform speeds without the re-drawing of video memory. The presence of a 12-bit A/D converter internal to the PGS allows a dynamic change in stimulus position, speed or pattern based upon physiological feedback. A physically realistic image size (0.9 cm2) and resolution (20 mu/pixel) in the retinal plane are achieved with simple intervening optics. The video field rate of 60 Hz is above the flicker fusion frequency for most vertebrate animals and does not induce artifacts in cellular responses. The PGS operating in a PC-based environment meets the requirements of a versatile optical stimulator for investigations in retinal electrophysiology.

Spatial resolution of images reconstructed from a bulk-detection scanning-laser microscope.

We present theory and experimental data on the minimum detectable feature size and spatial resolution for a scanning-laser microscope system that uses bulk photodetection. In the analysis, interactions of laser photons with an object are given a probability function that varies with the position within an object. Typical interactions that can be measured with such a scanning device include photon absorption (densitometry), scattering, and photofluorescence. Because bulk photodetection is used, image resolution is a function of laser-probe spot size and recording precision. We present data from simulations that predict a minimum separation between feature centers of approximately 1.56 times the half-width of the laser spot. Experimental verification by scans of U.S. Air Force test targets confirms this theory.

Collection and handling of ultrathin serial sections for 3-dimensional reconstruction.

Serial sectioning for 3-D reconstruction requires a highly skilled and experienced individual to collect ribbons of ultrathin sections on formvar-coated grids, and to handle the grids after section collection. A simple method is described for placing ribbons in an orderly serial fashion on formvar-coated grids, by a microtomist with average experience. Prior to sectioning, a wax ledge is prepared on the sloping edge of a glass knife in order to support a formvar-coated grid held in a horizontal slot cut in the wax. After a ribbon is formed, the water in the trough is slowly withdrawn to allow the ribbon to settle on the grid. The grids are then placed in an easy-to-make plastic chamber so that the formvar does not get ruptured during drying. The chamber can also be used for staining and storage of grids thereafter. Approximately 4000 sections from mudpuppy retinal cells have been successfully collected using this method. Computer 3-D reconstruction of the individual cells has been done.

Homozygote for Huntington disease.

Four offspring of three different Huntington disease (HD) affected x affected matings were assessed by genetic linkage analysis for possible homozygosity. One individual was found to have a 95% likelihood of being an HD homozygote. The homozygote individual had an age at onset and symptoms which were similar to those of affected HD heterozygote relatives, including some with younger onset. This confirms the observation of Wexler et al. that in HD the homozygote is not more severely afflicted than the heterozygote.

Clustering of multiallele DNA markers near the Huntington's disease gene.

Five highly informative multiallele restriction fragment length polymorphisms (RFLPs) of value for preclinical diagnosis of Huntington's disease (HD) have been genetically characterized. One RFLP was uncovered by expansion of the D4S43 locus while three others are at D4S111 and D4S115, loci defined by NotI-linking clones. The final marker, D4S125, represents a recently discovered VNTR locus. All four loci map closer to the HD gene and to the telomere than D4S10, the original linked marker for HD. In combination with two multiallele RFLPs previously identified for D4S43 and another linked locus, D4S95, these five new multiallele markers will dramatically improve the speed and accuracy of predictive testing in HD, and increase its applicability by maximizing the chances of an informative test for anyone with appropriate family structure.

A method for collecting semithin epoxy serial sections for light microscopy and 3-D reconstruction.

In the three-dimensional reconstruction of neuronal structure, it is imperative that ribbons of semithin or ultrathin sections be obtained. Resin-embedded semithin sections display better structural details than paraffin-embedded sections. The cutting and collecting of long ribbons of resin-embedded semithin sections using a microtome, requires the use of large troughs on glass knives. A simple plastic trough has been described which facilitates the collection of ribbons directly onto a coverslip. As the ribbons are formed, they are floated on a coverslip. The water in the trough is slowly drained through a tubing which is attached to a syringe. The ribbons settle on the coverslip, which is easily removed and placed on a hotplate to dry the sections.