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Biomedical subjects

J M Johnson

Publications and source records attributed to J M Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1997

Glutamine biosensors for biotechnology applications, with suppression of the endogenous glutamate signal.

Glutamine membranes for amperometric measurements are described. The interference of the endogenous glutamate is greatly diminished by using a supplementary "anti-glutamate" layer consisting of immobilized glutamate oxidase and catalase on top of the glutamine-sensitive layer having co-immobilized glutaminase and glutamate oxidase. The use of polycarbonate membranes with different permeability characteristics for the control of the substrate's access to the enzyme layers is presented, as well as the effect of the density of the enzyme layer on the sensitivity of these membranes. The fabricated membranes have good operational stability (at least 5 days) and very good linearity (up to 10 mM glutamine). Using an appropriate choice of membranes and cross-linking conditions, membranes with good rejection of glutamate have been fabricated (less than 6% RSD for a 5 mM glutamine sample containing 5 mM glutamate as interferent). These membranes are suitable for monitoring of glutamine levels in mammalian cell cultures without the need of a separate measurement for glutamate.

Amino Acid Oxidoreductases↗
PubMed1997

Treatment of experimental autoimmune encephalomyelitis with genetically modified memory T cells.

The migratory properties of memory T cells provide a model vector system for site-specific delivery of therapeutic transgene factors to autoimmune inflammatory lesions. Lymph node cells from (SWRxSJL)F1 mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with a DNA construct that placed the anti-inflammatory cytokine interleukin-10 (IL-10) cDNA under control of an antigen-inducible IL-2 promoter region. Isolated T cell clones demonstrated antigen-inducible expression of transgene IL-10 and expressed cell surface markers consistent with the phenotype of normal memory T cells. Upon adoptive transfer, transfected T cell clones were able to inhibit onset of experimental autoimmune encephalomyelitis (EAE) and to treat EAE animals therapeutically after onset of neurologic signs. Semiquantitative immunocytochemistry showed a significant correlation between decreased demyelination and treatment with the transfected T cells. Taken together, these data indicate the autoreactive T cells can be genetically designed to produce therapeutic factors in an antigen-inducible manner resulting in a decreased severity of clinical and histological autoimmune demyelinating disease.

Animals↗
PubMed1997

NMR solution structure of the antitumor compound PT523 and NADPH in the ternary complex with human dihydrofolate reductase.

The antitumor compound PT523 [N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine] was found to have an inhibition constant (K(i)) of 0.35 +/- 0.10 pM against human dihydrofolate reductase (hDHFR), 15-fold lower than that of the classical antifolate drug methotrexate (MTX). The structure of PT523 bound to hDHFR and hDHFR-NADPH was investigated using multinuclear NMR techniques. NMR data indicate that the binary complex has two distinct conformations in solution which are in slow exchange and that the addition of NADPH stabilizes the ternary complex in a single bound state. Comparison of resonance assignments in the PT523 and MTX ternary complexes revealed that substantial protein chemical shift differences are limited to small regions of hDHFR tertiary structure. A restrained molecular dynamics and energy minimization protocol was performed for the hDHFR-PT523-NADPH complex, using 185 NOE restraints (33 intermolecular) to define the ligand-binding region. The positions of the pteridine and pABA rings of PT523 and the nicotinamide and ribose rings of NADPH are well defined in the solution structures (RMSD = 0.59 A) and are consistent with previously determined structures of DHFR complexes. The N(delta)-hemiphthaloyl-L-ornithine group of PT523 is less well defined, and the calculated model structures suggest the hemiphthaloyl ring may adopt more than one conformation in solution. Contacts between the hemiphthaloyl ring and hDHFR, which are not possible in the hDHFR-MTX-NADPH complex, may explain the greater inhibition potency of PT523.

Antineoplastic Agents↗
PubMed1997

The use of tertiary collimation for spinal irradiation with extended SSD electron fields.

PURPOSE: The spine can be treated with an electron beam when its maximum posterior depth is within the therapeutic range of electrons. Electron fields treated at extended source-to-surface distances (SSDs), however, have larger penumbras and narrower therapeutic isodose widths relative to those at the standard SSD of 100 cm. We investigated the use of tertiary collimation close to the patient surface for these fields to sharpen the penumbra, minimizing dose to normal tissue and maximizing target coverage. METHODS AND MATERIALS: Using film dosimetry in a polystyrene phantom, we measured the dose distribution for electron fields at extended SSD under varying collimation conditions. Beam penumbra and therapeutic width as a function of depth, SSD, applicator insert size, and tertiary collimator opening were determined. We also measured the dose distributions in the junction region for various gaps between x-ray fields and an electron field as used for craniospinal irradiation. RESULTS: Measurements show that tertiary collimation close to the skin surface reduces penumbra width (lateral distance between the 90 and 20% isodose lines) by 56% and increases therapeutic isodose width (lateral width of the 90% isodose curve) by 25% at a depth of dmax relative to standard collimation. These numbers change to 23 and 13%, respectively, at an average depth of the spine. When lateral brain and posterior spine fields are used to irradiate the entire craniospinal axis, tertiary collimation aids in reducing the volume of the hot spot in the junction region by as much as 10% without compromising target coverage. CONCLUSIONS: Tertiary collimation for extended SSD electron fields is preferable to standard collimation in order to minimize dose to normal tissue and increase target coverage. This technique can be applied to both spinal and craniospinal irradiation. Support structures for the tertiary blocking are needed because the weight of the lead is usually too great for placement on the skin.

Electrons↗
PubMed1997

A broad-spectrum antifungal from the marine sponge Hyrtios erecta.

Spongistatin 1, a macrocyclic lactone polyether from the marine sponge Hyrtios erecta, was fungicidal for a variety of opportunistic yeasts and filamentous fungi, including strains resistant to amphotericin B, ketoconazole and flucytosine. In broth macrodilution assays, MICs ranged from 0.195 to 12.5 microg/ml, and minimum fungicidal concentrations ranged from 3.12 to 25 microg/ml. Initial disk diffusion screens with six related macrocyclic lactone polyethers from H. erecta and Spirastrella spinispirulifera, revealed that these polyethers were also antifungal. The fungicidal activity of spongistatin 1 was confirmed in killing kinetics studies, where killing of Candida albicans and Cryptococcus neoformans occurred within 6 and 12 h, respectively. During the killing kinetics experiments, non-treated C. albicans maintained the yeast morphology. However, elongated forms resembling germ tubes were the predominant morphologic form in spongistatin 1-treated C. albicans cultures. The spongistatins show promise as potential antifungal agents and as probes to study fungal morphogenesis and nuclear division.

Animals↗
PubMed1997

Evidence of functional beta-adrenoceptors in the cutaneous vasculature.

During a hyperthermic challenge, skin blood flow (SkBF) increases primarily through activation of the cutaneous active vasodilator system. However, mechanisms through which activation of this system elevates SkBF remain unknown. In this project, we sought to identify whether functional beta-adrenoceptors exist on cutaneous vessels and, if present, whether these receptors play an important role in elevating SkBF during a hyperthermic challenge. In protocol 1, SkBF was assessed over two intradermal microdialysis probes. Initially, both probes were perfused with lactated Ringer solution. Probe A was then perfused with a 200 microM solution of the beta-adrenoceptor agonist isoproterenol while probe B was perfused with a 1.7 mM solution of the beta-adrenoceptor antagonist propranolol. Isoproterenol perfusion significantly increased SkBF from 17.7 +/- 2.4 to 70.8 +/- 13.2 perfusion units (PU; P < 0.05), whereas propranolol perfusion did not significantly affect SkBF (23.4 +/- 6.5 to 27.0 +/- 6.8 PU; P > 0.05). After this period, the solutions perfusing the probes were switched. Isoproterenol did not significantly change SkBF at the propranolol-treated site (27.0 +/- 6.8 to 26.4 +/- 7.5 PU; P < 0.05). In protocol 2, SkBF was assessed over two microdialysis probes during indirect whole body heating. One probe was perfused with Ringer solution while the other probe was perfused with 1.7 mM propranolol. The degree of elevation in SkBF during heat stress at the propranolol-treated site (10.4 +/- 1.5 to 35.8 +/- 3.1 PU) was similar to the elevation in SkBF at the Ringer solution site (11.6 +/- 1.0 to 35.0 +/- 1.2 PU). These data demonstrate the presence of functional beta-adrenoceptors in the skin; however, these receptors play no significant role in mediating cutaneous vasodilation during indirect whole body heating.

Adrenergic beta-Agonists↗
PubMed1997

Modification of active cutaneous vasodilation by oral contraceptive hormones.

It is not clear whether the altered thermoregulatory reflex control of the cutaneous circulation seen among phases of the menstrual cycle also occurs with the synthetic estrogen and progesterone in oral contraceptive pills and whether any such modifications include altered control of the cutaneous active vasodilator system. To address these questions, we conducted controlled whole body heating experiments in seven women at the end of the third week of hormone pills (HH) and at the end of the week of placebo/no pills (LH). A water-perfused suit was used to control body temperature. Laser Doppler flowmetry was used to monitor cutaneous blood flow at a control site and at a site at which noradrenergic vasoconstrictor control had been eliminated by iontophoresis of bretylium (BT), isolating the active cutaneous vasodilator system. The oral temperature (Tor) thresholds for cutaneous vasodilation were higher in HH at both control [37.09 +/- 0.12 vs. 36.83 +/- 0.07 degrees C (LH), P < 0.01] and BT-treated [37. 19 +/- 0.05 vs. 36.88 +/- 0.12 degrees C (LH), P < 0.01] sites. The Tor threshold for sweating was similarly shifted (HH: 37.15 +/- 0.11 degrees C vs. LH: 36.94 +/- 0.11 degrees C, P < 0.01). A rightward shift in the relationship of heart rate to Tor was seen in HH. The sensitivities (slopes of the responses vs. Tor) did not differ statistically between phases. The similar threshold shifts at control and BT-treated sites suggest that the hormones shift the function of the active vasodilator system to higher internal temperatures. The similarity of the shifts among thermoregulatory effectors suggests a centrally mediated action of these hormones.

Adult↗
PubMed1997

Rehabilitation of dogs with surgically treated cranial cruciate ligament-deficient stifles by use of electrical stimulation of muscles.

OBJECTIVE: To determine effect of electrical muscle stimulation (EMS) on rate and degree of return to function of the limb and development of degenerative joint disease (DJD) after surgical creation and subsequent stabilization of the cranial cruciate ligament (CrCL)-deficient stifle. ANIMALS: 12 clinically normal adult large (19.5 to 31.5 kg) dogs. PROCEDURE: Dogs were anesthetized, and the right CrCL was severed via arthrotomy, destabilizing the stifle. After 3 weeks, the stifle was surgically stabilized. Three weeks later, 6 dogs were subjected to an EMS treatment protocol for the thigh muscles. At 5, 9, 13, and 19 weeks after stifle destabilization, treated (n = 6) and control (n = 6) dogs were evaluated for return of stifle function. Gross and histologic evaluations of the stifles were performed at 19 weeks after stifle destabilization. RESULTS: Treated dogs had significantly (P = 0.001) better lameness score than did control dogs. There was less palpable crepitation of the stifle in treated dogs (P = 0.06); treated dogs also had significantly (P = 0.01) fewer radiographic signs of bone changes. Thigh circumference was significantly (P = 0.02) larger in treated dogs. There was less gross cartilage damage (P = 0.07) in the EMS-treated dogs, but more medial meniscal damage (P = 0.058, cranial pole; P = 0.051, caudal pole). CONCLUSIONS: Improved lameness scores, larger thigh circumference, and decreased radiographically apparent bony changes observed for the treated group of dogs support the hypothesis that dogs treated by EMS after surgical stabilization of the CrCL-deficient stifle had improved limb function, with less DJD, than did dogs treated with the currently accepted clinical protocol of cage rest and slow return to normal activity. However, results of force plate evaluation did not support the hypothesis. Increased meniscal damage in dogs treated by EMS may be cause for concern.

Animals↗
PubMed1996

Closed reduction and type-II external fixation of comminuted fractures of the radius and tibia in dogs: 23 cases (1990-1994).

OBJECTIVE: To describe a technique for closed reduction of and application of a type-II external fixator to comminuted fractures of the radius and tibia in dogs and to evaluate outcome of the technique in a series of client-owned dogs. DESIGN: Retrospective case series. ANIMALS: 23 dogs that underwent closed reduction of severely comminuted (> or = 5 fragments); mid-diaphyseal radial (n = 9); and tibial (n = 14) fractures and stabilization with a type-II external fixator. PROCEDURE: Radiographs were made postoperatively and every 4 to 6 weeks until the fixator was removed. The effect of type of fracture (open vs closed), type of pins (threaded vs smooth), and number of fixation pins on surgery time, time between surgery and development of bridging callus, and time between surgery and removal of the fixator was evaluated using one-sided Student's t-tests. RESULTS: 21 fractures healed after a single surgery. Two dogs with radial fractures required a second procedure because of complications. All fractures healed with the original fixation device in place. Mean time between surgery and the development of bridging callus was 11.4 weeks (range, 4 to 22 weeks), and mean time between surgery and fixator removal was 14.7 weeks (range, 4 to 27 weeks). Type of fracture, type of pins, and number of fixation pins did not have a significant effect on surgery time, time to development of bridging callus, or time to fixator removal. CLINICAL IMPLICATIONS: Closed reduction and application of a type-II external fixator was an effective method of treating severely comminuted radial and tibial fractures.

Animals↗
PubMed1996

Generation of autonomously pathogenic neo-autoreactive Th1 cells during the development of the determinant spreading cascade in murine autoimmune encephalomyelitis.

Chronic progression of autoimmune disease is accompanied by the acquisition of autoreactivity to new self-determinants. Recent evidence indicates that this process, commonly referred to as determinant spreading, may be pathogenic for chronicity. Our studies on experimental autoimmune encephalomyelitis (EAE), a murine model widely used in multiple sclerosis (MS) studies, have shown that determinant spreading develops as a predictable sequential cascade of neo-autoimmunity during progression to chronic disease. By 7-8 weeks after immunization of (SWR x SJL)F1 mice with the immunodominant myelin proteolipid protein determinant (PLP 139-151), splenocytes consistently respond to the immunodominant myelin basic protein determinant (MBP 87-99). In the present study, we directly address the pathogenicity of neo-autoimmunity resulting from endogenous self-priming during the course of disease. Our results indicate that T cells responding to the spreading MBP 87-99 determinant produce a proinflammatory cytokine profile consistent with type 1 helper T cells (Th1) cells. In addition, splenocytes activated to the spreading MBP 87-99 determinant consistently transfer acute EAE in naive recipients even when T cells reactive to the priming PLP 139-151 immunogen are eliminated by bromodeoxyuridine (BUdR)-mediated photolysis. Our data indicate that endogenous neo-autoantigen priming during chronic autoimmune disease generates type 1 helper T cells (Th1) cells that are autonomously pathogenic. These results provide further evidence supporting the view that determinant spreading is a pathogenic process that leads to chronic progression of autoimmune disease.

Adoptive Transfer↗
PubMed1996

A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease.

The development of autoimmune disease is accompanied by the acquired recognition of new self-determinants, a process commonly referred to as determinant spreading. In this study, we addressed the question of whether determinant spreading is pathogenic for progression of chronic-relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis (MS). Our approach involved a systematic epitope mapping of responses to myelin proteolipid protein (PLP) as well as assaying responses to known encephalitogenic determinants of myelin basic protein (MBP 87-89) and myelin oligodendrocyte glycoprotein (MOG 92-106) at various times after induction of EAE in (SWR X SJL)F1 mice immunized with PLP 139-151. We found that the order in which new determinants are recognized during the course of disease follows a predictable sequential pattern. At monthly intervals after immunization with p139-151, responses to PLP 249-273, MBP 87-99, and PLP 137-198 were sequentially accumulated in al mice examined. Three lines of evidence showed that determinant spreading is pathogenic for disease progression: (a) spreading determinants mediate passive transfer of acute EAE in naive (SWR X SJL)F1 recipients; (b) an invariant relationship exists between the development of relapse/progression and the spreading of recognition to new immunodominant encephalitogenic determinants; and (c) after EAE onset, the induction of peptide-specific tolerance to spreading but not to nonspreading encephalitogenic determinants prevents subsequent progression of EAE. Thus, the predictability of acquired self-determinant recognition provides a basis for sequential determinant-specific therapeutic intervention after onset of the autoimmune disease process.

Amino Acid Sequence↗
PubMed1996

Prenatal diagnosis of uniparental disomy 15 following trisomy 15 mosaicism.

Maternal uniparental disomy 15 (UPD15), responsible for approximately 25 per cent of Prader-Willi syndrome cases, is usually caused by maternal meiosis I non-disjunction associated with advanced maternal age. These cases may initially be detected as mosaic trisomy 15 during routine prenatal diagnostic studies. In such cases, PCR (polymerase chain reaction) microsatellite analysis of uncultured cells makes prospective prenatal diagnosis for UPD15 possible with results available in 2-4 days. We have performed molecular analyses on a series of seven cases of mosaic trisomy 15 identified in amniotic fluid (AF, n = 3) or chorionic villus samples (CVS, n = 4) from patients initially referred for advanced maternal age or abnormal triple screen. In all cases, the maternal ages were > or = 35 years and maternal meiosis I non-disjunction was documented as the cause of the trisomy in all informative cases (n = 5). Of the three case with mosaic trisomy 15 at amniocentesis, two showed the presence of the trisomy in the fetus. Molecular analysis showed one case with maternal UPD15 in the euploid cell line and one case with biparental inheritance. Both of these families elected to terminate the pregnancies based on the presence of true fetal mosaicism. In the third case, low-level trisomy 15 mosaicism in the amniotic fluid was not confirmed in a follow-up amniotic fluid sample and molecular analysis indicated biparental inheritance in the fetus. For the four trisomy 15 mosaics detected at CVS, molecular analysis was performed on direct amniotic fluid cell lysates for prospective diagnosis of UPD at 14-16 weeks' gestation. Follow-up cytogenetic analysis of the amniotic fluid in all four cases was normal, indicating confined placental mosaicism. Molecular analysis showed one of these four cases to have maternal heterodisomy 15. Based on the likelihood of Prader-Willi syndrome due to maternal UPD15, the couple chose to terminate the pregnancy. The total of two of seven cases of trisomy 15 mosaicism resulting in UPD15 is consistent with the theoretical expectation of one-third and indicates a high risk of UPD in such pregnancies. Therefore, UPD testing should be offered in all cases of mosaic trisomy 15 encountered in CVS or amniocentesis.

Adult↗
PubMed1996

Improved dose distribution with a universal acrylic breast compensator.

Wedge filters frequently do not provide optimal distribution in the chest wall, lung, and areolar-nipple complex because they extend through the entire width of tangential radiation fields for breast cancer. In addition, medial wedge filters increase dose to the contralateral breast. In order to overcome these shortcomings, a new method of acrylic compensation was investigated. A universal acrylic breast compensator (ABC) was designed, with patient data, to extend anteriorly from the central axis. To accurately display build-up data, the ABC was entered into the treatment planning system as a wedge filter. Dose homogeneity within the compensated breast, chest wall, and lung volumes was assessed using 3-D planning and dose volume histograms. Dose to the contralateral breast was evaluated using thermoluminescent dosimetry in a polystyrene phantom. Opened, wedged, and compensated conditions were irradiated using the above described techniques. Due to lack of extension of the ABC posteriorly to the central axis (allowing lung inhomogeneity to correct for increased patient thickness), ABCs reduced the high dose regions in the chest wall and lung as compared to wedge filters. ABCs reduced hot spot in the areolar-nipple complex more efficiently as compared to wedge filters. ABCs also reduced dose to the contralateral breast by more than 20% relative to wedge filters. Wedge filters can be inefficient because of lung transmission and contour topography. Excessive dose to the chest wall, lung, and areolar-nipple complex, and contralateral breast can be alleviated with the use of a universal acrylic breast compensator. Its convenience and dosimetric advantages both in treatment planning and daily application offer a useful tool in the management of breast cancer.

Acrylic Resins↗