Biomedical subjects

M K Stevens

Publications and source records attributed to M K Stevens.

7 recordsLinked to original sources

PubMed1992

Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.

Adhesins, Bacterial↗

PubMed1991

Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.

Antibodies, Bacterial↗

PubMed1990

Systematic studies on the effects of the NMDA receptor antagonist MK-801 on cerebral blood flow and responsivity, EEG, and blood-brain barrier following complete reversible cerebral ischemia.

The dose-dependent effects of MK-801, a glutamate receptor antagonist, on changes in CBF, CBF-PaCO2 responsiveness (133Xe clearance), EEG, and blood-brain barrier (methylene blue) were examined after a 15-min period of reversible complete global ischemia induced in halothane-anesthetized cats by occlusion of the vertebral and carotid arteries. Pretreatment with doses of MK-801 of greater than or equal to 0.5 mg/kg had no effect on resting CBF measures and produced a dose-dependent slowing of the dominant EEG frequency. In animals receiving this agent, there was an almost immediate return of baseline EEG patterns upon reinstitution of flow, no hypoperfusion after 2 h of reflow, preservation of CBF and CBF-PaCO2 responsiveness, and maintenance of blood-brain barrier integrity. In contrast, parallel control animals and animals treated with MK-801 at a dose of 0.1 mg/kg exhibited poor recovery based on the above parameters. MK-801 also diminished in a dose-dependent manner the CSF levels of 6-keto-prostaglandin (PG) F1 alpha (stable metabolite of PGI2) and thromboxane (Tx) B2 (stable metabolite of TxA2), which were otherwise elevated in vehicle-treated animals 2 h after reflow. Of particular interest, the CSF TxB2/6-keto-PGF1 alpha ratio in vehicle-treated animals was near 2. In animals pretreated with MK-801, at doses of greater than or equal to 0.5 mg/kg, this ratio was nearly 1. These observations are consistent with a possible triggering role of glutamate release in initiating at least part of the acute sequelae of ischemia. Such release in an electrically silent cell would increase Ca2+ influx and activate free fatty acid metabolism, leading to probable changes in vascular function and changes in blood-brain barrier permeability.

Animals↗

PubMed1990

Disulfide-linked protein associated with Mycoplasma pneumoniae cytadherence phase variation.

Wild-type Mycoplasma pneumoniae possessed a protein with a very high molecular weight under nonreducing conditions (greater than 340,000; designated HMW5); this protein was absent from a noncytadhering phase variant lacking HMW1, 2, 3, and 4. When examined by two-dimensional nonreducing-reducing gel electrophoresis, HMW5 dissociated to yield a single polypeptide spot of molecular weight 190,000 that comigrated with cytadherence phase-variable protein HMW2. Extraction of wild-type mycoplasmas with Triton X-100 revealed the exclusive partitioning of HMW5 with the detergent-insoluble cytoskeletonlike triton shell.

Antigens, Bacterial↗

PubMed1988

Time course of release in vivo of PGE2, PGF2 alpha, 6-keto-PGF1 alpha, and TxB2 into the brain extracellular space after 15 min of complete global ischemia in the presence and absence of cyclooxygenase inhibition.

The time-dependent release of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), thromboxane (Tx) B2, and 6-keto-PGF1 alpha (6-keto) from brain was measured before, during, and after a 15-min interval of total ischemia (four-vessel occlusion) in halothane-anesthetized cats using the technique of cerebroventricular perfusion. Resting levels of PGE2, PGF2 alpha, 6-keto, and Tx were: 253 +/- 75, 953 +/- 300, 650 +/- 200, and 550 +/- 170 pg/ml, respectively. During the 15-min ischemia, all prostanoids rose significantly, yet the highest levels were not observed until the first 15-60 min of the reflow at which time levels of PGE2, PGF2 alpha, 6-keto, and Tx, as compared with the preischemic baseline, rose approximately 8, 3.4, 3, and 55-fold, respectively. Significantly, although all prostanoids showed increases relative to baseline, the ratios of PGF2 alpha/6-keto and PGE2/6-keto remained stable throughout the experiment in both groups of animals. In contrast, the Tx/6-keto ratio rose from approximately 1 to approximately 30 during the 60 min after reflow in untreated cats. Treatment with zomepirac sodium (5 mg/kg, i.v.), a cyclooxygenase inhibitor, resulted in highly significant reductions in the levels of all prostanoids during the preischemic period. In zomepirac sodium-treated animals, there were also highly significant reductions in the prostanoid response to ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗

PubMed1986

Effect of preischemia cyclooxygenase inhibition by zomepirac sodium on reflow, cerebral autoregulation, and EEG recovery in the cat after global ischemia.

Zomepirac sodium (ZS) (5 mg/kg i.v.) was used to evaluate the effects of preischemia cyclooxygenase inhibition on CBF (as assessed by 133Xe clearance), CBF-PaCO2 responsiveness, and electrophysiologic (EEG) parameters before and after a 15-min period of complete global ischemia produced by four-vessel occlusion and mild hypotension. During the 15-min period of ischemia, CBF was essentially zero. Following reflow all groups displayed an initial hyperemia as compared with control (92 +/- 11 vs. 141-146 ml/100 g/min). Saline-treated animals during reflow displayed a delayed hypoperfusion (26 +/- 3 ml/100 g/min), which showed no improvement during the 2-h reflow period prior to death. In contrast, ZS-treated animals during reflow displayed significantly higher flows during the hypoperfusion phase (72 +/- 9 ml/100 g/min). The CBF-PaCO2 response displayed an approximately sevenfold reduction in slope at 2 h after reflow in saline-treated animals. This decrease in PaCO2 reactivity was not observed in the ZS-pretreated animals. With regard to EEG, all animals showed a total flattening during the 15 min of ischemia. In saline-treated animals only one of seven showed any sign of even marginal recovery. In ZS-treated animals EEG activity showed prominent recovery in seven of seven. Brainstem auditory evoked potentials were monitored and showed prominent recovery of amplitude and latency in ZS but not saline-treated animals during reflow.

Animals↗

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