Biomedical subjects
Publications and source records attributed to S M Johnson.
The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals with Coccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge with C. immitis.
Proteins secreted into the culture medium by Mycobacterium tuberculosis are thought to play an important role in the development of protective immune responses. In this report, we describe the molecular cloning of a novel, low-molecular-weight antigen (MTB12) secreted by M. tuberculosis. Sequence analysis of the MTB12 gene indicates that the protein is initially synthesized as a 16.6-kDa precursor protein containing a 48-amino-acid hydrophobic leader sequence. The mature, fully processed form of MTB12 protein found in culture filtrates has a molecular mass of 12. 5 kDa. MTB12 protein constitutes a major component of the M. tuberculosis culture supernatant and appears to be at least as abundant as several other well-characterized culture filtrate proteins, including members of the 85B complex. MTB12 is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex, the BCG strain of M. bovis, and M. leprae. Recombinant MTB12 containing an N-terminal six-histidine tag was expressed in Escherichia coli and purified by affinity chromatography. Recombinant MTB12 protein elicited in vitro proliferative responses from the peripheral blood mononuclear cells of a number of purified protein derivative-positive (PPD+) human donors but not from PPD- donors.
An in vitro brain stem preparation from adult turtles (Chrysemys picta) was used to examine the effects of anoxia and increased temperature and pH/CO2 on respiration-related motor output. At pH approximately 7.45, hypoglossal (XII) nerve roots produced patterns of rhythmic bursts (peaks) of discharge (O.74 +/- 0.07 peaks/min 10.0 +/- 0.6 s duration) that were quantitatively similar to literature reports of respiratory activity in conscious, vagotomized turtles. Respiratory discharge was stable for 6 h at 22 degrees C; at 32 degrees C, peak amplitude and frequency progressively and reversibly decreased with time. Two hours of hypoxia had no effect on respiratory discharge. Acutely increasing bath temperature from 22 to 32 degrees C decreased episode and peak duration and increased peak frequency. Changes in pH/CO2 increased peak frequency from zero at pH 8.00-8.10 to maxima of 0.81 +/- 0.01 and 1.44 +/- 0.02 peaks/min at 22 degrees C (pH 7.32) and 32 degrees C (pH 7.46), respectively; pH/CO2 sensitivity was similar at both temperatures. We conclude that 1) insensitivity to hypoxia indicates that rhythmic discharge does not reflect gasping behavior, 2) increased temperature alters respiratory discharge, and 3) central pH/CO2 sensitivity is unaffected by temperature in this preparation (i.e., Q10 approximately 1.0).
An in vitro brain stem preparation from adult turtles was used to determine effects of dopamine (DA) and norepinephrine (NE) on the pattern of respiratory motor output recorded from hypoglossal nerve roots (XII). Bath-applied DA (10-200 microM) increased the frequency of respiratory bursts (peaks) from 0.9 +/- 0.2 to 2.4 +/- 0.3 (SE) peaks/min, resulting in a 99 +/- 9% increase in neural minute activity. R[+]-SCH-23390 (10 microM, D1 antagonist) and eticlopride (20 microM, D2 antagonist) attenuated the DA-mediated increase in peak frequency by 52 and 59%, respectively. On the other hand, the DA-receptor agonists apomorphine (D1, D2), quinelorane (D2), and SKF-38393 (D1) had no effect on peak frequency. Prazosin, an alpha1-adrenergic antagonist (250 nM) abolished the DA-mediated frequency increase. Although NE (10-200 microM) and phenylephrine (10-200 microM, alpha1-adrenergic agonist) increased peak frequency from 0.5 +/- 0.1 to 1.2 +/- 0.3 peaks/min and from 0.6 +/- 0.1 to 1. 0 +/- 0.2 peaks/min, respectively, these effects were not as large as that with DA alone. The data suggest that both dopaminergic and adrenergic receptor activation in the brain stem increase respiratory frequency in turtles, but the DA receptor-mediated increase is dependent on coactivation of alpha1-adrenergic receptors.
The authors have been professionally and personally associated with osteopathic medicine since 1972. During this period, they have observed, from several perspectives, the processes by which trainees and osteopathic physicians inculcate their unique professional identity. Yet, increasingly, the philosophic and practical components that have historically defined osteopathic medicine as a distinctive approach to medical practice are rapidly eroding. Powerful forces associated with such things as professional prestige, public acceptance, professional collaboration with allopathic physicians, as well as changing trainee expectations, are rapidly reshaping the osteopathic medical profession. The degree to which osteopathic medical practitioners embrace the philosophic and clinical components unique to their profession will determine whether the profession retains its identity as a separate medical entity. If the current de-emphasis of these identifying characteristics continues, little more than a name will distinguish osteopathic medicine from the allopathic medical profession.
BACKGROUND: Hepatic artery thrombosis (HAT) remains a devastating complication after liver transplantation. Various factors have been implicated in the pathogenesis of HAT, such as clotting abnormalities, increased hematocrit, and technical complications, but the role of anticardiolipin antibodies has not been evaluated. We investigated the possible association between HAT and anticardiolipin antibodies in adult patients who underwent liver transplantation. METHODS: Seven patients with HAT after orthotopic liver transplantation, 28 liver recipients without HAT, and 35 normal blood donors were evaluated. Determination of IgM and IgG anticardiolipin antibodies was performed by enzyme-linked immunosorbent assay using pretransplant serum from all allograft recipients. Clinical information was obtained from chart review. Fisher's exact test and Wilcoxon rank sum test were used for statistical analysis, and all P-values were two-tailed. RESULTS: Overall, 22 of 35 (63%) liver recipients had a positive anticardiolipin antibody test (either IgG or IgM titer >4 SD from the normal controls). The test was positive in 7 liver recipients (100%) with HAT compared with 15 out of 28 patients (54%) without HAT (P=0.031). As compared with liver recipients without HAT, patients with HAT also tended to have a higher mean anticardiolipin titer of IgG and IgM and a lower pretransplant platelet count; however, these differences were not significant. CONCLUSIONS: Our findings indicate that anticardiolipin antibodies are frequently elevated in patients with liver failure and may contribute to the pathogenesis of HAT after liver transplantation. Other potential consequences of anticardiolipin antibodies in end-stage liver disease remain to be determined.
The spontaneously hypertensive (SH) rat has an exaggerated sympathetic discharge which may result from an enhanced neuronal excitability in the central nervous system. To test this hypothesis, we examined the electrophysiological properties of neurons in the medial region of the nucleus of the solitary tract (mNTS), a central nucleus involved in the processing of baroreceptor afferent information, in SH rats and normotensive Sprague-Dawley (SD) rats. An in vitro brainstem slice preparation was used to record intracellularly from 18 neurons in 4-5-month-old SH rats and 16 neurons in 4-5-month-old SD rats. Between the two groups there was no significant differences in resting membrane potential, input resistance, and spontaneous firing frequency, or in action potential amplitude, duration, and after-hyperpolarization (AHP). There were no significant differences in spike frequency adaptation and post-tetanic hyperpolarization (PTH). Delayed excitation (DE), a manifestation of A-current, occurred in 88% in SH and SD mNTS neurons, but the duration of DE was significantly (P < 0.05) shorter in SH mNTS neurons. We propose that attenuated expression of A-current may contribute to increased sympathetic drive in SH rats.
Excitatory amino acids (EAAs) have been implicated as mediators of cell death in neurodegenerative diseases involving catecholamine neurons. Few studies, however, have examined the toxic effects of EAAs on identified catecholamine neurons in vitro. We have investigated the neurotoxic effects of kainic acid in a rat brain substantia nigra (SN) slice preparation. Rats (60-80 g) were anesthetised with halothane and killed by cervical dislocation. SN slices, 300 microm thick, were incubated at 35 degrees C in a modified Krebs solution in the presence or absence of kainic acid and then fixed and processed for either immunohistochemistry (IHC) or electron microscopy (EM). In IHC experiments, SN neurons were labeled using antibody to tyrosine hydroxylase (TH) coupled to diaminobenzidine. In control slices, the antibody labeled not only the cell body but also the prolific dendritic arbor of SN neurons. Treatment with 50 microM kainic acid for 15 min or 2 h resulted in loss of TH staining and apparent fragmentation of the dendrites. EM provided ultrastructural evidence for kainic acid-induced degeneration of the dendritic arbor of SN neurons. Typically, the dendritic membrane was broken, or diffuse and collapsed. Ultrastructural damage, including clumping and marginalization of chromatin and vacuolation of the cytoplasm, was also observed in cell bodies. Damage to the dendritic arbor may occur early in the neurotoxic events leading to cell death, preceding the loss of the cell body. Our observations are consistent with the postulated role of EAAs as mediators of catecholamine neuron death.
BACKGROUND: Our purpose was to determine whether cyclodialysis clefts can be imaged with magnetic resonance imaging (MRI). METHODS: Surgical cyclodialysis clefts extending approximately 3 clock hours were created in four New Zealand white rabbits. Eyes were scanned with an ocular MRI coil. Images obtained after intravenous gadolinium, topical godalinium, and gadolinium injected into the cleft were compared to images obtained without contrast. Two human eyes were also scanned for cyclodialysis clefts with MRI. RESULTS: Direct injection of gadolinium into the suprachoroid space yielded definitive localization and delineation of the cyclodialysis cleft. Cyclodialysis clefts could also be imaged following enhancement with topical or intravenous gadolinium. Without contrast medium, the clefts could not be clearly identified in rabbits. In a patient with hypotony and choroidal effusion following cataract surgery, a cyclodialysis cleft and enhancement of the suprachoroidal space were found with intravenous administration of gadolinium. MRI from a patient with a trabeculo-suprachoroidal shunt also demonstrated gadolinium enhancement of the suprachoroidal space. CONCLUSION: Cyclodialysis clefts can be imaged using gadolinium-enhanced MRI in rabbits and humans.
This study examined client variables expected to predict success in emotionally focused marital therapy (EFT), now the second most validated form of marital therapy after the behavioral approaches. The relationship of attachment quality, level of emotional self-disclosure, level of interpersonal trust, and traditionality to the therapy outcome variables, marital adjustment, intimacy, and therapist ratings of improvement, was examined. These variables were chosen for their relevance to the theory and practice of EFT and to intimate relationships in general. Overall, therapeutic alliance predicted successful outcome; the task dimension of the alliance in particular predicted couples' satisfaction. More specifically, one dimension of female partners' trust, their faith in their partner, predicted couples' satisfaction at follow-up. Females' faith also significantly predicted males' level of intimacy at follow-up. Males who were most likely to be nondistressed at termination indicated higher levels of proximity seeking on an attachment measure at intake, and older males and males whose partners had higher levels of faith in them were more likely to be nondistressed at follow-up. Traditionality was not found to be significantly related to outcome. Couples who made the most gains at follow-up also indicated lower initial marital satisfaction and included males who indicated lower levels of use of attachment figure on the attachment measure at intake. Males who made the largest gains at termination were older and were rated as less expressive by their partner on self-disclosure measures at intake. Age was the only variable significantly related to males' gains in satisfaction at follow-up. Implications for the practice of marital therapy and future research are delineated.
N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission is implicated in activity-dependent developmental reorganization in mammalian brain, including sensory systems and spinal motoneuron circuits. During normal development, synaptic interactions important in activity-dependent modification of neuronal circuits may be driven spontaneously (Shatz 1990b). The respiratory system exhibits substantial spontaneous activity in utero; this activity may be critical in assuring essential and appropriate breathing movements from birth. We tested the hypothesis that NMDA receptors are necessary for prenatal development of central neural circuits underlying respiratory rhythm generation by comparing the responsiveness of control mice and mutant mice lacking the NMDA receptor R1 subunit (NMDAR1) gene to glutamate receptor agonists and antagonists and comparing endogenous respiratory-related oscillations generated in vitro by brain stem-spinal cord and medullary slice preparations from control and mutant mice. In control mice, local application of NMDA and the non-NMDA receptor agonist, (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA), over the pre-Bötzinger Complex, the C4 cervical motor neuron pool, and the hypoglossal motor nucleus produced profound increases in inspiratory frequency, tonic discharge on C4 ventral nerve roots, and inward currents in inspiratory hypoglossal motoneurons, respectively. Responses of mutant mice to AMPA were similar. However, mutant mice were completely unresponsive to NMDA applications. Preparations from mutant mice generated a respiratory rhythm virtually identical to control. Results demonstrate that NMDA receptors are not essential for respiratory rhythm generation or drive transmission in the neonate. More importantly, they suggest that NMDA receptors are not obligatory for the prenatal development of circuits producing respiratory rhythm.
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A questionnaire was mailed to 2000 randomly selected osteopathic physicians to assess use of osteopathic manipulative treatment (OMT). In all, 1055 responses were summarized for the study. The contention is supported that OMT is being used less and less by practicing physicians. Only 6% of the respondents treated more than 50% of their patients with OMT, and nearly one third used OMT on less than 5% of their patients. A progressive de-emphasis of OMT use correlated with more recent graduation from osteopathic medical colleges. Thirty-eight percent of the variance regarding OMT use was attributed to two factors: barriers to use, and OMT protocol used. Perceptions by physicians of insufficient OMT training were not predictive of decreased use of OMT. Significantly more OMT was used in solo practice as opposed to other settings. The results present a wake-up call for the osteopathic medical profession. The profession must strive to remove barriers that preclude OMT use by justifying to policymakers, health professionals, and the public the cost-benefits of OMT in holistic healthcare.
The Breast Imaging Reporting and Database System (BI-RADS) was developed by the American College of Radiology and is used by a number of computerized mammography tracking systems. The ability of BI-RADS to encode the data contained in 300 mammography reports at the Columbia-Presbyterian Medical Center was examined. BI-RADS was able to encode normal reports and "special masses" (such as lymph nodes) without difficulty. However, none of the general masses and only 17% of the calcifications could be encoded in BI-RADS. The implications of this for the design of mammography databases are discussed.
The physiological characteristics of central neural populations are being increasingly explored in slice preparations. A major challenge of this approach is to correlate the physiological properties of individual neurones or groups of neurones with their anatomical and chemical properties in order to gain key insights into their functional identities. The present study describes a method for determining the precise topographical position and the immunohistochemical characteristics of neurones in brain slice preparations that are used frequently in electrophysiological investigations. Thick horizontal slices of rat brainstem were re-cut using a method that provided thin sections that were always in the same plane as the parent slice and that were of suitable thickness for immunohistochemistry. Catecholaminergic neurones in these co-planar (horizontal) sections were stained using antisera to tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. To identify individual catecholamine neurones in the co-planar sections, we constructed a reference atlas of the distribution of catecholamine neurones in the horizontal plane of the rat brain. The combined use of the horizontal atlas and of immunohistochemical techniques in co-planar sections of horizontal slices enables the determination of several key properties: (1) whether a neurone is TH-positive, (2) its precise topographical position and (3) its content of neuropeptides and other immunohistochemical markers. Thus our study offers a readily feasible method for correlative anatomy and immunohistochemistry of physiologically identified catecholaminergic neurones in brain slices.
A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation.
The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli. The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies. The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody. The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID. The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin. The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera. Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.
Slice preparations from neonatal rat medulla that generate respiratory rhythm in vitro were used to test for Gi/o protein-mediated mechanisms affecting breathing rhythm in mammals. The frequency of inspiratory motor discharge recorded from hypoglossal (XII) nerve roots decreased with bath application of gamma-aminobutyric acid (GABA) and norepinephrine, as well as agonists specific for GABAB, alpha 2-adrenergic, and mu-opioid receptors; 5-hydroxytryptamine had little effect on frequency. Microinjection of these specific agonists into the pre-Bötzinger complex, the site of respiratory rhythm generation in vitro, also decreased frequency. In contrast, substance P (SP) increased frequency when it was bath applied or microinjected into the pre-Bötzinger complex. To test for involvement of Gi/o proteins, pertussis toxin (PTX) was injected into the cerebrospinal fluid of newborn rats, and slices from these animals were tested 48 h later for block of drug effects on rhythm. In PTX-treated slices the frequency decrease due to GABAB, mu-opioid, and alpha 2-adrenergic receptor activation was attenuated (P < or = 0.05), whereas the SP receptor-mediated response was unaltered. To test for involvement of K+ conductances linked to Gi/o proteins Ba2+ (0.2 mM) was added to the bath before application of drugs. Ba2+ attenuated the decrease in frequency associated with GABAB (P < or = 0.05) and mu-opioid (0.10 < or = P < or = 0.05) receptor activation, whereas the alpha 2-adrenergic and SP responses were unaltered. We conclude that GABAB and mu-opioid, but not alpha 2-adrenergic and SP, receptor activation modulates respiratory frequency via a Gi/o protein-dependent Ba(2+)-sensitive ionic conductance mechanism on neurons within the medullary locus for rhythm generation. This mechanism may be a convergent pathway for control of respiratory frequency.