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Biomedical subjects

R Knowles

Publications and source records attributed to R Knowles.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nitric oxide synthase is expressed in human macrophages during foreign body inflammation.

Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes.

Adult↗
PubMed1996

Hypoxemia/reoxygenation down-regulates interleukin-8-stimulated bactericidal activity of polymorphonuclear neutrophil by differential regulation of CD16 and CD35 mRNA expression.

BACKGROUND: The purpose of this study was to determine the effects of hypoxemia/reoxygenation (H/R) on the regulation of interleukin-8 (IL-8)-stimulated human polymorphonuclear neutrophil (PMN) bactericidal activity. METHODS: Venous human whole blood was rendered normoxic (Pvo2 saturation 60% to 80%), hypoxemic (Pvo2 saturation, less than 15%), or H/R (Pvo2 saturation more than 97%) by dialyzing the blood against a gas mixture of N2/H2/CO2 +/- 30% O2. Two hundred microliter aliquots from each study group were incubated with IL-8 (50 ng/ml) for 45 minutes before fluorescein isothiocyanate-conjugated mouse antihuman CD16 or CD35 antibodies were added. Bactericidal activity was measured with the release of 51Cr from labeled bacteria at 1:1, 5:1, and 10:1 PMN-target ratios. Steady-state mRNA levels for CD16 and CD35 were quantified by Northern blot analyses. RESULTS: H/R reduced PMN bactericidal activity compared with hypoxemic levels for staphylococcus aureus (48 +/- 5.6 versus 27 +/- 3.3) and Escherichia coli (58 +/- 7.1 versus 33 +/- 4.2). H/R reduced the surface expression of CD16 but not CD35 (mean channel fluorescence CD16, 610 +/- 70 versus 310 +/- 30 for hypoxemia versus H/R; p < 0.01). After H/R was performed, IL-8 decreased mRNA levels for CD16 but not for CD35 compared with levels seen during hypoxemia + IL-8. CONCLUSIONS: H/R down-regulates IL-8-stimulated PMN bactericidal activity by decreasing steady-state mRNA levels and surface expression of CD16. PMN bactericidal capability after H/R + IL-8 is primarily complementary and not Fc gamma receptor dependent.

Blood Bactericidal Activity↗
PubMed1996

Standardization of pain management in the postanesthesia care unit.

A project is described in which a standard in the Post Anesthesia Care Unit (PACU) for managing patients with acute pain was implemented. The nurses' documentation and their perceptions concerning pain management were assessed. The data collected from the pre-questionnaires showed misconceptions about pain medication, lack of knowledge about measurement tools, and value systems that were inconsistent with recognizing a comfortable and safe "comfort level" of pain for patients. The pre-audit of PACU nursing documents showed that the assessment of pain was noted in terms of presence or absence but not quantified by the use of a measurement standard. After the implementation of a pain standard, results of the post-questionnaires showed changes in behaviors, attitudes, and knowledge that showed significant increases in the use of a numerical or visual pain tool, and an increase in documented evaluation of pain.

Health Knowledge, Attitudes, Practice↗
PubMed1996

Nitric oxide synthase activity in malaria-infected mice.

Nitric oxide (NO) is implicated in a variety of major cellular functions including defence from invasion by microbical pathogens. Evidence has been presented suggesting that it is an important mediator of protection in the early non-specific responses to malaria in mice infected with Plasmodium chabaudi (Taylor-Robinson et al. 1993). Other data from in vitro studies on the asexual stages of human parasite Plasmodium falciparum indicated that while nitric oxide itself may not be inhibitory to parasite development, its downstream products do have some anti-plasmodial activity (Rockett et al. 1991) and these could be generated by macrophages (Gyan et al. 1994). Similarly, the sexual phases of both rodent (Motard et al. 1993) and human malaria (Naotunne et al. 1993) are reportedly susceptible to the toxic effects mediated by nitric oxide generated by blood leucocytes in the course of transmission to the mosquito vector.

Animals↗
PubMed1996

Expression of recombinant parvovirus NS1 protein by a baculovirus and application to serologic testing of rodents.

A recombinant baculovirus containing the NS1 gene of minute virus of mice was constructed. Optimal expression of the recombinant NS1 protein (rNS1) was achieved by infecting Trichoplusa ni High Five cells at a multiplicity of 10 and incubating them for 72 h postinfection. An enzyme-linked immunosorbent assay (ELISA) with rNS1 as the antigen was evaluated for serologic testing of laboratory rodents. The rNS1 ELISA proved to be a more sensitive method for the detection of antibodies to recently recognized rodent parvovirus species (mouse orphan parvovirus and rat orphan parvovirus) and prototypic parvovirus species (minute virus of mice, Kilham's rat virus, and H-1) than were conventional parvovirus ELISAs that use whole parvovirus virions.

Amino Acid Sequence↗
PubMed1996

Denitrification: microbiology and ecology.

The ability of some aerobic microorganisms to reduce nitrogen oxides, especially nitrate, to gaseous products enables them to grow in oxygen-limiting conditions by using the oxides as electron acceptors. Denitrifying respiration is not as efficient as oxygen respiration so growth is slower. The ability to denitrify is found in a wide variety of unrelated bacteria and even in some fungi, and biochemical mechanisms are now moderately well understood. Major factors regulating denitrification are the availability of the nitrogen oxides, the availability of reductant (mostly organic carbon compounds but inorganic compounds may be used), and decreased oxygen concentration. These three factors are in turn governed by many other factors such as water content, pH, porosity, and the presence of inhibitory compounds, which may act to cause accumulation of ionic (nitrite) or gaseous (nitric oxide, nitrous oxide) intermediates. The prediction of rates of denitrification and release of intermediates such as nitrous oxide in particular environments is proving to be difficult.

Bradyrhizobiaceae↗
PubMed1995

Hypoxemia up-regulates interleukin-8 stimulated phagocytosis of polymorphonuclear leukocytes by differential regulation of CD32w and CD35 messenger RNA expression.

BACKGROUND: The purpose of these studies was to investigate the effect of hypoxemia on interleukin-8 (IL-8) regulation of phagocytosis and surface opsonic receptor expression of whole blood polymorphonuclear leukocytes (PMNs). METHODS: Whole blood PMNs were rendered hypoxemic (PvO2 less than 15 mm Hg) or normoxic (PvO2 equals 60 to 80 mm Hg) and incubated with IL-8 (50 ng/ml) before the sequential addition of serum-opsonized fluorescent microspheres (emission wavelength, 560 nm) and fluorescein isothiocyanate-conjugated mouse anti-human CD32w or CD35 antibodies (emission wavelength, 513 nm). Concomitant two color flow cytometric analysis was then performed to measure the mean channel fluorescence (MCF) and the percentage of positive PMNs. Steady-state messenger RNA levels for CD32w and CD35 were quantitated by means of Northern blot analysis by using total RNA extracted from purified PMNs from each study group. RESULTS: Hypoxemia increased IL-8 stimulated PMN phagocytosis (percentage of positive PMNs, 38 +/- 1.5 versus 27 +/- 2.0, MCF, 5491 +/- 182 versus 4060 +/- 121 for hypoxemia plus IL-8 versus normoxia plus IL-8, respectively; p < 0.05). Hypoxemia increased IL-8 stimulated PMN expression of CD32w and CD35 (MCF CD32w, 791 +/- 105 versus 336 +/- 81; MCF CD35, 542 +/- 87 versus 254 +/- 41; p < 0.05). Under normoxic conditions IL-8 decreased messenger RNA levels for both CD32w and CD35, but under hypoxemic conditions IL-8 increased messenger RNA levels only for CD32w. CONCLUSIONS: Hypoxemia directly regulates IL-8 control of PMNs by increasing phagocytosis, receptor expression, and messenger RNA for these receptors. Studies investigating cytokine regulation of PMN function need to take into account oxygen tension when establishing the fundamental effects of cytokines on PMN physiology.

Cytokines↗
PubMed1995

Hyperacuity test to evaluate vision through dense cataracts; research preliminary to a clinical study. I. Studies conducted at the University of California at Berkeley before travel to India.

BACKGROUND. Patients with dense ocular media disorders retain the ability to project or point to an intense source of light. Using this response capability and high luminance points of light as stimuli, Vernier judgments (a hyperacuity test) can be made by these patients, even without the presence of a "window" through a leucoma, cataract, or bleed. Without coaching, these individuals are able to locate the centers of the individual degraded point images if the individual light sources are adequately separated (i.e., if sufficient "gaps" exist between the individual stimuli), and they can spatially align the degraded images. Advanced cataracts are the main cause of blindness in the developing world, and this is a treatable condition. In these nations, only a modest proportion of affected patients receive surgery, and only 5% or less of these individuals obtain treatment in two eyes. There are incredibly large and rapidly growing backlogs of advanced cataract patients requiring care (many millions). Because of the 20 to 30% failure rates that occur after treatment (all causes) in many developing world settings, a test performed before surgery, which offers a meaningful estimate of postsurgical visual outcome, can be valuable. Using the principle defined above, we seek to determine before surgery those individuals who will derive most benefit from cataract removal, and which of two cataractous eyes has the better postsurgical visual prognosis. EXPERIMENTAL. In Berkeley, we performed a series of preliminary studies on a Vernier acuity test before initiating a clinical study in a developing world setting. These studies were conducted upon young adult normal subjects wearing their usual vision corrections, with and without induced refractive errors, and/or with or without simulated dense nuclear cataracts. We sought (1) to determine the number of repeat trials necessary for reliable outcomes; (2) to compare a two-point and a three-point Vernier acuity display; (3) to determine the shape of the measured response function at large gap separations between test points; (4) to define optimal test distance and stimulus size; (5) to assess the effect(s) of a broad range of uncorrected refractive errors upon outcomes; and (6) to consider means to minimize refraction-based errors by using a pinhole, a refractive correction, and/or selective spatial filtering. We compared responses obtained using the current CRT/VDT-based, computer-driven (Berkeley) instrument with a new precision optical/mechanical computer-driven (India) instrument. The India instrument is needed to determine design parameters for a next stage simpler, cheaper, more rugged field instrument(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1995

Hyperacuity test to evaluate vision through dense cataracts; research preliminary to a clinical study. II. Initial trials of the India instrument and HASP protocol at Aravind Eye Hospital, Madurai, India.

BACKGROUND. Our purpose is to develop a clinical test capable of assessing vision through dense cataracts (and other advanced media opacities) before surgery. Because such anomalies are endemic in the developing world, it was desirable to conduct a clinical study of a prototype India instrument and HyperAcuity Study Protocol (HASP) in such a setting. This investigation, preliminary to a planned clinical study, was conducted at the Aravind (Free) Eye Hospital, Madurai, India. EXPERIMENTAL. The preliminary HASP protocol, developed in Berkeley, was adapted to existing clinical practices in Madurai. Included was a new test of visual acuity, the Gap "VA" test, developed as part of the revised protocol. The rational for this test, methods used, and sample data are presented. A Vernier alignment test (one of the hyperacuities) is used to assess vision through dense ocular media disorders. The task of the patient is to align vertically three high luminance, discrete visual stimuli. The revised HASP protocol and adapted India instrument were tested upon patients with advanced cataracts. The cataract grading system used in these studies (provided by Drs. N. V. Projna and G. Rohini) is defined. Patients were tested before and shortly after surgery. This investigation allowed us to refine the test protocol and instrument design preparatory for a clinical study of HASP. A much simpler/cheaper version of the India instrument will be used in the planned clinical study. Additional experiments are scheduled in order to address issues which require resolution before initiation of next-stage testing. In part, in these papers, we seek to help others understand some problems encountered when conducting research in a developing world environment, even a very good one!

Aged↗
PubMed1995

Performance on three-point vernier acuity targets as a function of age.

With certain (but not all) vernier acuity (VeA) stimulus displays observers exhibit no or slight performance decrements with aging. Because three-point VeA test displays are much more easily applied experimentally and clinically, we examined visual performance on this task as a function of age. Ages of groups tested ranged from 20 to 70+ years (some individuals were 90+ years old). Eyes were examined monocularly. We tested three- and two-point sharply defined VeA arrays and a three-point ground-glass-degraded display (simulating a 20/100 nuclear cataract). Gap or feature separations were varied. Results obtained with the three-point display showed little alteration with age. Image degradation resulted in reduction in performance when small gap separations were used between the test points, but only slight decrements for large gap separations were recorded. The finding that performance with certain VeA targets is largely unaffected by age is confirmed and is expanded.

Adult↗
PubMed1995

Perioperative oxygen saturation levels of pediatric patients.

Airway compromise is of immediate concern for any patient after surgery but more so for pediatric patients because they are subject to more acute airway changes than adults. The purpose of this study was to identify those pediatric post-surgical patients in an acute care setting at risk for oxygen saturation less than 90% during transport from the OR to the PACU. A descriptive, correlational design was employed using a convenience sample of 45 male and female pediatric patients between the ages of 0 months and 16 years who underwent elective surgery with general anesthesia lasting longer than 1 hour. Data were collected on each pediatric subject at four points in time (1) in the preoperative holding area (2) in the OR, (3) during transport to the PACU, and (4) on arrival to and during the stay in the PACU. Results showed that more than 50% of the sample (N = 45) desaturated to oxygen levels below 95%. Specifically, 48% desaturated between 90% to 95% and 8% desaturated below 90%. Pediatric patients age 2 years and younger showed the greatest risk for desaturation below 90%. In addition, as transport time increased, the number of subjects with oxygen saturation levels below 95% increased.

Adolescent↗
PubMed1994

Organization of actin and microtubules during process formation in tau-expressing Sf9 cells.

Insect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau-expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau-induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation.

Actin Cytoskeleton↗
PubMed1994

Cellular regulation of nitrate uptake in denitrifying Flexibacter canadensis.

Nitrate uptake and its regulation were investigated using an ion-specific nitrate electrode for denitrifying Flexibacter canadensis under anaerobic conditions. Glucose supported a greater rate of nitrate uptake than did glycerol, glutamate, lactose, cellobiose, or ethanol. Nitrate uptake closely approximated Michaelis--Menten kinetics; the estimated Ks(glucose) and apparent Km(nitrate) for nitrate uptake were 21 and 44 microM, respectively. Nitrate disappearance was correlated with nitrite accumulation, and nitrate had an inhibitory effect on nitrite reduction. Oxygen inhibition of nitrate uptake increased as the percent air saturation increased, and reversed readily as the percent air saturation decreased. The minimal air saturation showing inhibition of nitrate uptake was about 2-4%. Azide and cyanide completely inhibited nitrate uptake. No nitrate uptake was observed in cells grown in the presence of 1 or 5 mM tungstate (no added molybdate). When molybdate (100-200 microM) was present in the medium, nitrate uptake was exhibited by organisms grown with 1 mM, but not with 5 mM, tungstate, indicating that nitrate uptake was dependent on the presence of an active nitrate reductase, and that competition between tungsten and molybdenum occurred during the formation of nitrate reductase. Nitrite production from nitrate by whole cells but not cell-free extracts was inhibited by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, indicating that nitrate and (or) nitrite transport depended upon the electrochemical proton gradient.

Bacteroidetes↗
PubMed1994

Beta-adrenergic receptor-mediated regulation of extracellular adenosine in cerebral cortex in culture.

Adenosine is an important inhibitory neuromodulator in the CNS, yet the sources of extracellular adenosine have yet to be well characterized. In this study we show that beta-adrenergic stimulation of cortical cultures results in the extracellular accumulation of cAMP as well as adenosine, and that the extracellular adenosine derives from extracellular cAMP. The concentration dependence of isoproterenol in evoking cAMP secretion was determined by radioimmunoassay, and the EC50 for this effect was found to be approximately 100 nM. In order to investigate the effect of beta-adrenergic stimulation on the regulation of extracellular adenosine, the effect of isoproterenol in stimulating the extracellular accumulation of adenine-containing compounds was examined by HPLC. Isoproterenol stimulated cAMP secretion in both astrocyte cultures and astrocyte-rich mixed cultures of astrocytes and neurons. However, no extracellular cAMP was detectable in neuron-enriched astrocyte-poor cultures. Extracellular adenosine increased in response to isoproterenol in the astrocyte-rich mixed cultures, but not in the neuron-enriched astrocyte-poor cultures. After 30 min exposure to isoproterenol, the concentration of adenosine in the extracellular medium increased by 47% in 56 experiments in the mixed astrocyte-rich cultures. In order to establish whether the adenosine that accumulates in response to isoproterenol stimulation actually derives from extracellular cAMP, phosphodiesterase inhibitors were tested for their ability to block isoproterenol-stimulated adenosine accumulation. Isobutylmethylxanthine (IBMX; 100 microM), RO 20-1724 (180 microM), carbazeran (10 microM), dipyridamole (10 microM), and trifluoperazine (10 microM) had no inhibitory effect on the isoproterenol-stimulated accumulation of extracellular adenosine. However 100 microM IBMX plus 180 microM RO 20-1724 effectively blocked isoproterenol-stimulated adenosine accumulation and, as expected, increased extracellular cAMP. As a further test of the origin of isoproterenol-stimulated adenosine accumulation, we attempted to block this phenomenon by blocking cAMP secretion itself. For this purpose probenecid, a known inhibitor of cAMP secretion in many different cell types, was used. We found that probenecid at 1 mM blocked isoproterenol-stimulated adenosine accumulation. These studies suggest that one potentially important source of extracellular adenosine in the cerebral cortex is endogenous extracellular cAMP, secreted from astrocytes in response to beta-adrenergic receptor stimulation. Since the receptors of neuromodulators other than norepinephrine may also be coupled to adenylyl cyclase in the cerebral cortex, there may be several neuromodulatory systems that regulate extracellular adenosine levels by this mechanism.

Adenine Nucleotides↗
PubMed1993

Conservation of the amino acid sequence of SV2, a transmembrane transporter in synaptic vesicles and endocrine cells.

SV2 is a secretory vesicle-specific protein produced by all neurons and by endocrine cells. The deduced amino acid (aa) sequence of this protein indicates that it is a transmembrane transporter [Bajjalieh et al., Science 257 (1992) 1271-1273; Feany et al., Cell 70 (1992) 861-867; Gingrich et al., FEBS Lett. 312 (1992) 115-122]. To determine the regions of the protein that are the most highly conserved throughout evolution, and might therefore be essential for the function of SV2, we isolated a cDNA clone encoding SV2 from the elasmobranch fish, Discopyge ommata, and compared the deduced aa sequence to two isoforms from rat, SV2A and SV2B [Bajjalieh et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2150-2154]. The comparison indicates that although the N-terminal cytoplasmic domain of SV2 is the most divergent region, it contains a highly conserved sequence that is predicted to be the epitope for a monoclonal antibody that crossreacts with all species and two isoforms of SV2 [Buckley and Kelly, J. Cell Biol. 100 (1985) 1284-1294; Bajjalieh et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2150-2154]. The remainder of the protein is highly conserved: 62% of the aa in SV2 from D. ommata are identical to the rat SV2A sequence, and 12% are conservative substitutions. The high degree of conservation of this protein throughout evolution and across species indicates that it mediates a critical function of synaptic vesicles.

Amino Acid Sequence↗
PubMed1993

Chlorpromazine inhibits both the constitutive nitric oxide synthase and the induction of nitric oxide synthase after LPS challenge.

The effects of chlorpromazine on either the activity of mouse brain nitric oxide synthase or the induction of lung nitric oxide synthase in mice and rats were studied. Chlorpromazine inhibited the nitric oxide synthase activity in mouse brain cytosol. This effect could be reversed by adding an excess of calmodulin. In addition, chlorpromazine was able to inhibit the induction of lung nitric oxide synthase, in both species, after LPS administration. Furthermore, chlorpromazine also inhibited arginase activity in mouse lung cytosol.

Amino Acid Oxidoreductases↗