Muscular urinary sphincter: electrically stimulated myoplasty for functional sphincter reconstruction

PURPOSE: To reconstruct an electrically stimulated muscular urinary sphincter (MUS) using a tailored gracilis muscle free flap with intact nerve. MATERIALS AND METHODS: Unilateral surgically tailored gracilis muscle free flaps were transferred into the pelvis in eight dogs, leaving the obturator nerve intact. The muscle's pedicle vessels were anastomosed to the inferior epigastric artery and vein in the pelvis and the muscle was wrapped around the bladder neck. Electrodes were inserted into the MUS and connected to a programmable pulse generator. After 8 weeks of training the MUS, the pulse generator was programmed to be "on" for 4 hours and "off' for 15 minutes in a continuous cycle. Urodynamic studies were performed periodically, and at the end of the experiment the MUS and proximal urethra were harvested for histology. Three control dogs had sham operations. RESULTS: All MUS's functioned well following the procedure. Histology of the MUS/urethra complex showed no evidence of stricture. Except for one dog, all urethras were easily catheterized. CONCLUSIONS: This electrically stimulated innervated free-flap MUS technique effectively increases bladder outlet resistance without producing urethral obstruction.     

Saltatory propagation of Ca2+ waves by Ca2+ sparks

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.     

Flow-cytometric analysis of peripheral blood lymphocytes in patients with chronic idiopathic neutropenia of adults

Blood traces tortuous paths of flow through the heart. We postulate that momentum changes associated with direction changes optimize dynamic coupling between ventricular and atrial function, particularly on exercise. Traces of pulsed Doppler mitral flow and M-mode long-axis mitral ring movement were recorded before and during exercise, increased in 25-W steps to strenuous levels (146 +/- 30 W), in 16 healthy volunteers, aged 38 +/- 10 years. R-R intervals fell from 821 +/- 151 to 437 +/- 51 ms, and diastole from 458 +/- 134 to 169 +/- 33 ms. Peak mitral flow velocities rose from 0.68 +/- 0.17 to 1.27 +/- 0.16 m/s, and mitral valve ring displacements from 13.8 +/- 3.3 to 19.3 +/- 3.4 mm. Biphasic diastolic curves of flow and movement became monophasic as R-R fell below 500 ms, with atrial systole apparently coming to coincide with elastic ventricular recoil to give a single elevated peak of mitral flow. The increased slope and amplitude of Doppler curves indicate increased rates of change of momentum, which imply enhanced inertial forces. The illustrated patterns of flow and movement on exercise accord with the postulated "dynamic" mode of function, in which forces between atria, ventricles, and passing blood masses become tightly coupled to achieve a sling-like redirection of momentum through tortuous paths of flow, but more extensive data are needed to adequately model and quantify inertial force exchanges of the exercising heart.     

Duration of Borrelia burgdorferi infectivity in white-footed mice for the tick vector Ixodes scapularis under laboratory and field conditions in Ontario

The duration of Borrelia burgdorferi infectivity in white-footed mice (Peromyscus leucopus) experimentally inoculated or infested with infected Ixodes scapularis nymphs was evaluated. Infectivity was assessed by infesting these mice with unfed I. scapularis larvae at 7, 21, 35 and 49 days post-inoculation (DPI) or post-infestation (PI). At 7 DPI, B. burgdorferi was transmitted from 18 of 24 syringe-inoculated mice and all three tick-infected mice to I. scapularis larvae which fed upon them. However, at 21, 35 and 49 DPI, significantly fewer mice were infective. Borrelia burgdorferi was isolated from tissues of 14 of 22 syringe-inoculated mice about 56 DPI, and from all three tick-infected mice. However, the level of agreement between xenodiagnosis and bacterial culture was no greater than would be expected by chance alone. We also determined if B. burgdorferi infectivity of mice varied in relation to periods of tick feeding in the field. White-footed mice were trapped during April, July and August 1993 from two habitats on Long Point peninsula (Ontario, Canada), where B. burgdorferi is endemic. Mice from each habitat were infested with laboratory-reared I. scapularis larvae. Ticks from each mouse were subsequently examined by immunofluorescent assay for B. burgdorferi infection and mice were cultured for B. burgdorferi. None of 3577 I. scapularis larvae fed on 62 mice captured within the cottonwood dune habitat were infected with B. burgdorferi, although it was isolated from six of these mice. Within the maple forest habitat, 0/24, 8/21 (38%) and 1/21 (5%) mice transmitted B. burgdorferi to I. scapularis larvae during April, July and August, respectively. Most mice from the maple forest with B. burgdorferi-positive tissues (14/21) were collected during July, although the level of agreement between xenodiagnosis and tissue culture was poor. Because B. burgdorferi infectivity in mice appears to be of short duration, overwintered I. scapularis larvae and nymphs may have to feed upon infected hosts at the same time of year in order for a cycle of B. burgdorferi infection to be maintained on Long Point. Infected I. scapularis nymphs, rather than persistently infected vertebrate hosts, likely serve as the overwintering "reservoir" for B. burgdorferi on Long Point.     

Intraoperative determination of small intestinal viability following ischemic injury: a prospective, controlled trial of two adjuvant methods (Doppler and fluorescein) compared with standard clinical judgment

Two adjuvant techniques for the intraoperative assessment of small intestinal viability were compared with standard clinical judgment in a prospective, controlled study of 71 ischemic bowel segments in 28 consecutive patients operated on for acute intestinal ischemic disease. Each segment was independently assessed 15 minutes after surgical correction of the underlying lesion by: 1) standard clinical judgment; 2) Doppler-detected pulsatile mural blood flow; and 3) fluorescein ultraviolet fluorescence pattern. Viability endpoint for each segment was determined objectively by patient follow-up or "blinded" microscopic evaluation of histologically unequivocal resection specimens using criteria established by previous animal studies. Seventeen histologically equivocal specimens were excluded from the final results. Standard clinical judgment proved moderately accurate overall (89%) but would have led to a relatively high rate (46%) of unnecessary bowel resection. The Doppler technique did not increase accuracy in any category of evaluation. The fluorescein fluorescent pattern was correct in all 54 determinant bowel segments, and proved more sensitive specific, predictive, and significantly more accurate overall than either standard clinical judgment or the Doppler method. This controlled study suggests that the fluorescein technique is the method of choice for the prediction of small intestinal recovery following ischemic injury.     

Treatment of complex anterior urethral stricture disease with mesh graft urethroplasty

In this report we describe the toxicokinetics of the Tylenol Extended Relief (TER) preparation of acetaminophen in human overdose. We collected 41 cases of TER overdose from five regional poison centers. Patients who met the following criteria were studied: a single ingestion of TER alone; confirmed time of ingestion; at least four acetaminophen determinations; and normal concentrations of liver function enzymes. With the exception of standard decontamination measures, treatment with N-acetylcysteine (NAC) if any acetaminophen level was above the treatment line of the Rumack-Matthew nomogram, and additional acetaminophen determinations, no interventions were recommended. Our study group comprised 13 patients, 12 female and 1 male, with single overdoses of 10.4 to 65 g TER. The acetaminophen elimination half-life was 3.1 +/- .8 hours (mean +/- SD; range, 1.3 to 4.0 hours; n = 12). The elimination phase for patients 2, 3, 4, 6, 8, 9, 11, 13 was delayed until 8.0 +/- 2.8 hours (range, 5 to 14 hours) after ingestion. Patients 3, 8, and 11--who had initial acetaminophen levels below the "possible toxicity" line of the Rumack-Matthew nomogram--later had acetaminophen levels above this line. No patient demonstrated a late or second acetaminophen peak. We conclude that the elimination half-life of TER acetaminophen is similar to that reported in overdose of immediate-release acetaminophen overdose. In a subgroup of patients, drug absorption continued beyond the 2 to 4 hours previously reported in immediate-release acetaminophen overdose. On the basis of our data, the use of a single 4-hour acetaminophen determination may lead to failure to recognize patients with potentially toxic TER ingestion. Until more toxicokinetic data are available, a reasonable approach would be to obtain at least one additional acetaminophen determination at least 4 to 6 hours after the first, if the first is obtained 4 to 8 hours after ingestion. NAC treatment should be initiated if either level is above the nomogram line but not if both levels fall below the nomogram line.     

Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophus W3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca2+ and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cis peptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold beta-propeller, each "blade" of which is a four-stranded antiparallel twisted beta-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four beta-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold beta-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight beta-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the beta-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent beta-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca2+ is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.     

Reproducibility of measurements of potential doubling time of tumour cells in the multicentre National Cancer Institute protocol T92-0045

We compared the flow cytometric measurement and analysis of the potential doubling time (Tpot) between three centres involved in the National Cancer Institute (NCI) protocol T92-0045. The primary purpose was to understand and minimize the variation within the measurement. A total of 102 specimens were selected at random from patients entered into the trial. Samples were prepared, stained, run and analysed in each centre and a single set of data analysed by all three centres. Analysis of the disc data set revealed that the measurement of labelling index (LI) was robust and reproducible. The estimation of duration of S-phase (T(S)) was subject to errors of profile interpretation, particularly DNA ploidy status, and analysis. The LI dominated the variation in Tpot such that the level of final agreement, after removal of outliers and ploidy agreement, reached correlation coefficients of 0.9. The sample data showed poor agreement within each of the components of the measurement. There was some improvement when ploidy was in agreement, but correlation coefficients failed to exceed values of 0.5 for Tpot. The data suggest that observer-associated analysis of T(S) and tissue processing and tumour heterogeneity were the major causes of variability in the Tpot measurement. The first two aspects can be standardized and minimized, but heterogeneity will remain a problem with biopsy techniques.     

Gene expression of cellular retinoid-binding proteins: modulation by retinoic acid and dexamethasone in postnatal rat lung

Nicotine is reported to have toxic effects on gonadal functions, in addition to its established role in the pathogenesis of atherosclerosis and lung cancer. So nicotine-induced biochemical changes were studied in liver and testes. Chronic administration of nicotine was found to produce enhanced synthesis of cholesterol, triglycerides, phospholipids and free fatty acids in the liver and testes. The activity of the lipogenic enzymes was high in liver but unaltered in testes. The testosterone and estradiol levels in the serum were lower. As the changes brought about by chronic administration of nicotine were counteracted by mecamylamine, a known inhibitor of nicotine, it was proven that nicotine is having a specific gonadotoxic effect.