Biomedical subjects
Publications and source records attributed to E A Johnson.
A patient presented with near complete airway obstruction due to a massive tumor. Nonsurgical methods failed to secure the airway, and surgical approaches were considered unlikely to succeed in a timely fashion. Cardiopulmonary bypass via femoral-femoral cannulation with the use of local anesthesia and a portable unit, followed by IV anesthesia, allowed the surgeons to perform a controlled tracheotomy.
Carotenoids have recently received considerable interest because of their potential in delaying or preventing degenerative diseases such as arteriosclerosis, cancer, and aging. In this study we show that the active oxygen species singlet oxygen (1O2) and peroxyl radicals differently affect carotenoid composition and biosynthesis in the yeast Phaffia rhodozyma. Photochemical generation of 1O2 with rose bengal or alpha-terthienyl induced carotenoid accumulation. In contrast, peroxyl radicals derived from t-butylhydroperoxide (tBOOH) or H2O2 decreased the content of astaxanthin and increased beta-carotene by approximately 4-fold, suggesting end product feedback regulation by astaxanthin or inhibition of biosynthetic enzymes. 14C labeling of carotenoids during oxidative stress supported the possibility of end product regulation. Carotenoids were bleached by 8 mM tBOOH within 6 h when carotenogenesis was inhibited by thymol. When treated with peroxides, a previously unreported pigment in P. rhodozyma was formed. The carotenoid had a mass of 580 Da and a molecular formula of C40H52O3. Chemical derivatizations combined with mass and absorbance spectroscopy tentatively identified the carotenoid as dehydroflexixanthin (3,1'-dihydroxy-2,3,3',4'-tetradehydro-1',2'-dihydro-beta,psi-caro tene-4-one). This study provides the first report of induction of astaxanthin biosynthesis by 1O2, probable feedback control by astaxanthin, and the oxidative degradation of astaxanthin to novel pigments in P. rhodozyma.
Corticotropin-releasing factor (CRF) is a neuropeptide found throughout the central nervous system that has a proposed role in modulating emotional and behavioral states, including stress and anxiety. The amygdala, which is important in the control of emotional and autonomic responses to stress, contains CRF nerve terminals, CRF cell bodies, and CRF receptors. In rats, exposure to prenatal stress results in offspring that display a hyperemotional state and increased anxiety. In this study the effects of prenatal stress on CRF release was measured in amygdala minces (1 mm3) obtained from adult (8-16 weeks of age) male offspring of dams subjected to daily saline injection (0.1 ml, s.c.) from gestational day 14 to 21. CRF release from amygdala was time- and calcium-dependent, and stimulated by KCl-induced depolarization. Depolarization-induced CRF release was significantly increased by 42% from the amygdala of prenatally stressed offspring versus controls. Prenatally stressed offspring also showed a 49% increase in CRF levels in the amygdala. The increased amounts of CRF released in response to depolarization were likely the consequence of increased tissue content of CRF, as fractional release under basal or KCl-stimulated conditions was not different in the prenatal stress group versus control. This suggests that a long-lasting up-regulation of the CRFergic neurotransmission may occur in the amygdala, which may be important in the generation of hyperemotional offspring after exposure to prenatal stress.
Although the LD50 has been used to quantify the biologically active toxin in clinical preparations of botulinum A toxin (Botox and Dysport), a discrepancy exists between the clinical potency of equivalent international units of different formulations of botulinum A toxin for multiple clinical indications. Our laboratory previously reported that a regional chemodenervation assay in the mouse could be utilized to detect the difference in the potencies of the clinical preparations of toxin [Pearce et al. (1994) Toxic. appl. Pharmac. 128, 69-77]. The purpose of this study was to quantify the regional paralysis produced by botulinum toxin and define a new pharmacologic/biologic unit of activity that more accurately reflects the mechanism of action of botulinum toxin in the clinical setting. Quantal analysis of regional paralysis revealed that the ED50, defined as the median paralysis unit (MPU) for Botox and Dysport, was 0.41 +/- 0.01 and 1.00 +/- 0.02 LD50 units, respectively. Differences in the potencies found in retrospective clinical studies comparing Botox and Dysport were accurately reflected, for the first time, by the dose of toxin expressed in terms of the MPU (median paralysis unit). The data suggested that the MPU may be a more appropriate measure of the biologic activity in therapeutic formulations of botulinum toxin.
P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.
Escherichia coli O157:H7 was adapted to acid by culturing for one to two doublings at pH 5.0. Acid-adapted cells had an increased resistance to lactic acid, survived better than nonadapted cells during a sausage fermentation, and showed enhanced survival in shredded dry salami (pH 5.0) and apple cider (pH 3.4). Acid adaptation is important for the survival of E. coli O157:H7 in acidic foods and should be considered a prerequisite for inocula used in food challenge studies.
Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum.
Clostridium botulinum type G produces a toxin complex that is composed of neurotoxin, hemagglutinin, and nontoxic nonhemagglutinin. The three genes encoding these proteins were closely linked on a plasmid of about 114 kb (76 MDa) but not on chromosomal DNA. In contrast to the genes of other C. botulinum serotypes, the genes encoding type G toxin are on a plasmid.
Infection of young adult BALB/cByJ mice with mouse parvovirus-1, a newly recognized, lymphocytotropic, nonpathogenic parvovirus, was examined by in situ hybridization. Virus appeared to enter through the small intestine and was disseminated to the liver and lymphoid tissues. Strand-specific probes detected virion DNA in a consistently larger number of cells than replicative forms of viral DNA and/or viral mRNA. The number of signal-positive cells in the intestinal mucosa, lymph nodes, spleen, and thymus increased through day 10 after oral inoculation but decreased after seroconversion. Positive cells were still detected, however, in peripheral lymphoid tissues of mice examined at 9 weeks postinoculation. The results underscore the need to assess potential effects of persistent mouse parvovirus-1 infection on immune function in mice.
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In contrast to euthymic juvenile rats, which develop acute, self-limiting infection with rat virus (RV), RV infection of juvenile athymic rats was persistent for up to 12 weeks as demonstrated by recovery of infective virus, transmission to cagemates, and detection of viral DNA in the lungs. Administration of RV antiserum at the time of virus inoculation prevented persistent infection in five of six rats. Among rats given RV antiserum 1 week after virus, the interval at which euthymic rats begin to seroconvert, RV was not detected 1 week later but was recovered from four of six rats 3 weeks later. Results of these studies confirm that T-cell deficiency facilitates persistent RV infection and indicate that antibody provides significant protection from persistent infection only if it is present at the time of virus inoculation. The results support the concept that factors which prevent persistent infection in euthymic rats act early after virus inoculation and may include cellular immunity.
Similarly to other serotypes, botulinum neurotoxin serotype G (BoNT/G) contains the zinc binding motif of zinc endopeptidases. Highly purified preparations of BoNT/G show a zinc-dependent protease activity specific for VAMP/synaptobrevin, a membrane protein of synaptic vesicles. The two neuronal VAMP isoforms are cleaved with similar rates at one Ala-Ala peptide bond present in the same region, out of the several such peptide bonds present in their sequences. This site of cleavage is unique among the eight clostridial neurotoxins. VAMP proteolysis is displayed only after reduction of the single interchain disulfide bond present in the toxin, and it is inhibited by EDTA, o-phenanthroline and captopril.
The effect of variations in substitution on the conformation of iduronate-containing sequences in heparin and heparan sulphate has been studied using a series of chemically-modified heparins in which substitution with O- and N-sulphate and N-acetyl substituents has been systematically altered. Monosaccharides corresponding to residues in these modified heparins have also been investigated. The conformations of the glycosidic linkages in O- and N-desulphated re-N-acetylated heparin, O-desulphated re-N-sulphated heparin, and 6-O-desulphated re-N-sulphated heparin have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have previously been reported [B. Mulloy, M.J. Forster, C. Jones, and D.B. Davies, Biochem. J., 293 (1993) 849-858]. The overall conformation of all the polysaccharides is shown to be similar, regardless of substitution pattern. The conformational equilibrium of the pyranose ring of iduronic acid residues in the polysaccharides has been monitored by the use of 13C NMR chemical shift temperature coefficients, and shown to be similar for all the modified heparins with the exception of N-desulphated re-N-acetylated heparin. Circular dichroism spectra of all the polysaccharides are reported, and their variations attributed to differences in the proportions of pyranose ring forms in the iduronate conformational equilibrium.
Munchausen's syndrome is a chronic factitious disorder characterized by frequent hospitalizations, self-inflicted injuries, and dramatic medical histories. People with this condition assume the role of a sick patient and submit to unnecessary invasive, painful, and even dangerous medical procedures. In review of the literature, there have been four reports of patients feigning oncological disease. We admitted a 27-year-old woman who had undergone operative insertion of a Port-A-Cath and multiagent chemotherapy for "advanced ovarian cancer." Physicians should be aware of Munchausen's syndrome in order to avoid costly medical procedures and unnecessary operations and to stop the patient's vicious circle of pathological lying and self-inflicted injury.
Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods.
The public trusts hospitals for their diagnostic and therapeutic capabilities, while resentful of the high costs of care. A comprehensive national health care program will increase the public's distrust of health care when its goals are not met. It is more desirable to incrementally add benefits in a national program beginning with a catastrophic coverage program to assure the public's trust.
The fatty acid composition of membrane lipids from sarcolemma and sarcoplasmic reticulum isolated from biceps and gastrocnemius muscle has been compared in normal (wildtype, +/adrmto or +/+) and affected (adrmto/adrmto) myotonic mice. The adrmto mouse exhibits an arrested development of the righting response, and arose spontaneously from the SWR/J strain. These mice exhibit classical myotonia similar to the human disease, Becker's myotonia [1]. Significant alterations, characterized by a decrease in the saturated fatty acid, palmitic acid (16:0), and the polyunsaturated fatty acid, arachidonic acid (20:4), and an increase in stearic (18:0) and linoleic (18:2) acids, were observed between sarcolemma and sarcoplasmic reticulum from normal and affected mice. These changes in fatty acid composition of muscle membrane from ADR mice may be adequate to cause an alteration in membrane fluidity and affect the function of ion channels. The fatty acid composition of erythrocytes ghosts was also examined, as a potential marker for alterations in muscle membranes. In erythrocyte ghosts isolated from affected mice, the only alteration observed was a decrease in the proportion of oleic acid (18:1), an effect completely different from those observed in muscle membranes. Therefore, erythrocyte ghosts do not serve as an adequate indicator of changes in fatty acid composition of muscle membranes in this model of myotonia.
In situ hybridization and virus titration were used to characterize early stages of rat virus (RV) infection of rat pups after oronasal inoculation. Results suggest that virus enters through the lung and that early viremia leads rapidly to pantropic infection. Cells derived from all three germ layers were infected with RV, but those of endodermal and mesodermal origin were the predominant targets. Infection of vascular endothelium was widespread and was associated with hemorrhage and infarction in the brain. Convalescence from acute infection was accompanied by mononuclear cell infiltrates at sites containing RV DNA. Viral DNA was also detected in endothelium, fibroblasts and smooth muscle myofibers four weeks after inoculation. Further examination of these cells as potential sites of persistent infection is warranted.