Biomedical subjects
Publications and source records attributed to M G Davis.
An empirical method for verifying the total treatment time for either a one- or a two-catheter high-dose-rate procedure has been developed. The method can be performed quickly and allows for easy verification of the accuracy of the treatment time arrived at by a computerized planning system. The method is designed to confirm the treatment time to within 10%.
Recently, the mechanical failure of one of the upper collimator mechanical trimmers on a cobalt-60 unit resulted in large beam asymmetries and unacceptable flatness characteristics. This malfunction was not detected using currently accepted schedules for quality assurance tests. The incident suggests that the frequency of routine beam profile constancy checks should be increased to weekly for cobalt-60 units.
Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.
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Diabetic patients may experience fluctuations in whole blood glucose (WBG) levels while receiving hyperbaric oxygen therapy (HBO) resulting in seizure-like activity. Therefore, hyperbaric medical attendants must accurately monitor the WBG levels of these patients during HBO. In addressing this concern, this study evaluated the accuracy and reliability of commercially available glucometers (Glucometer M+, Companion 2, HemoCue, One Touch II, and ExacTech Pen) in the hyperbaric environment. WBG samples were prepared, ranging from 25 to 250 mg/dl, for testing glucometers at ground level and at 2.36 atmospheres absolute (ATA). It was noted that at 2.36 ATA, glucose values increased using the Glucometer M+, but decreased with the Companion 2 and HemoCue. The One Touch II values decreased in the hyperglycemic ranges (> 150 mg/dl), whereas the ExacTech Pen monitor readings increased in the hypoglycemic ranges (< 100 mg/dl). The accuracy of WBG monitors is significantly affected by changes in atmospheric pressure.
Event-related brain potentials (ERPs) were recorded from 6-month-old infants with and without Down syndrome presented with a visual recognition memory task. The ERP morphology was the same for both groups. The chronometry of information processing by infants with Down syndrome was similar to or faster than that of the infants without Down syndrome, depending on ERP component. The amplitude differences between groups may implicate frontal attentional processes in Down syndrome as opposed to more posterior processes. Infants with Down syndrome had an amplitude decrement in Nc over the central but not frontal cortex. The infants with Down syndrome also had similar visual fixation. Infants may have more subtle differences than those found in older individuals with Down syndrome.
Alpha-n1 interferon (Wellferon), alpha-2a interferon (Roferon), and alpha-2b interferon (Intron-A) inhibited accumulation of intracellular replicative forms of hepatitis B virus (HBV) in chronic producer cells by inhibiting accumulation of RNase-resistant HBV RNA. In contrast, the nucleoside analog FTC (cis-5-fluoro-1- [2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine) inhibited the accumulation of HBV DNA.
Platelet protein makeup is determined during transformation of megakaryoblasts to mature megakaryocytes, the immediate precursor of circulating platelets. To better understand the molecular mechanisms of megakaryocyte formation, gene expression was characterized by Northern analysis and RNA fingerprinting of cultured human CHRF-288 megakaryoblastic cells undergoing phorbol ester-stimulated megakaryocytic differentiation or serum-stimulated megakaryoblast proliferation. Protooncogenes c-fos and c-jun were coordinately upregulated in both proliferating and differentiating cells, whereas c-myc transcripts were upregulated during proliferation only. In contrast, mRNAs for transforming growth factor-beta 1 (TGF-beta 1) and thromboxane receptors were coordinately upregulated during differentiation but differentially regulated during proliferation. RNA fingerprinting revealed multiple transcripts specific to either proliferating or differentiated cells. Three of these were identified by homology to known DNA sequence: CDw44 adhesion molecule (upregulated during differentiation), glutathione sulfhydryl peroxidase (downregulated during differentiation), and plectin cytoskeletal protein (upregulated during differentiation). Thus, although megakaryoblast proliferation and megakaryocyte differentiation both involve DNA and protein synthesis, each growth response is characterized by a distinct pattern of gene expression.
The goal of this study was to determine the role of tyrosine phosphorylation in transducing deformation-stimulated vascular smooth muscle growth. Rat aorta-derived vascular smooth muscle cells were cultured on flexible silicone elastomer membranes and subjected to cyclic deformation (15 cycles per minute, deformed 2 seconds, relaxed 2 seconds). Deformation significantly increased proto-oncogene expression, [3H]thymidine incorporation, [3H]leucine incorporation, and cell number. Time course studies showed an 8-hour lag between initiation of cell deformation and onset of [3H]thymidine incorporation, with peak levels achieved after 18 to 24 hours. Western analysis of protein blots from deformed cells (10 minutes) demonstrated increased levels of phosphotyrosine-containing proteins having molecular weights of 110 to 130 and 70 to 80 kD. Deformation-stimulated tyrosine phosphorylation was prevented by the tyrosine kinase inhibitor Herbimycin A. Tyrosine kinase inhibition also prevented deformation-stimulated vascular smooth muscle cell growth as measured by [3H]thymidine incorporation. Cyclic deformation stimulates vascular smooth muscle proliferation through activation of tyrosine kinases. Inhibition of tyrosine phosphorylation is an effective means of preventing deformation-induced vascular smooth muscle growth in vitro.
Thromboxane A2 (TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC alpha, delta, epsilon, and zeta. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC alpha, delta, and epsilon by phorbol 12-myristate 13-acetate (PMA) but not TXA2 or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidine and increased cell number. Like TXA2 and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2 or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II--stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)
Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21-->Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax of 289,056 +/- 60,220 sites/cell and a Hill coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)
We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.
To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.
Necrotizing invasive pseudomonal infection of the external auditory canal (malignant external otitis) is an uncommon but important disorder in the elderly. The high morbidity, and even mortality, of this disorder has been reduced by the early and intensive use of combination antipseudomonal antibiotics. However, in severely immunocompromised patients or in infection involving the base of the skull, multiple cranial nerves, or the meninges, conventional therapy has been prolonged, intensive, and relatively ineffective. We treated 16 patients with malignant external otitis with adjuvant hyperbaric oxygen therapy. In six patients, infection was in advanced stages, infections were recurrences after previous treatment, and repeated treatment with antipseudomonal antibiotics had failed. All 16 cases responded promptly when a 30-day course of hyperbaric oxygen was added to the antibiotic regimen, and all patients remained free of infection or neurologic deficit during 1 to 4 years of follow-up. No complications of this treatment modality were noted. Hyperbaric oxygen therapy reverses tissue hypoxia, which enhances phagocytic killing of aerobic microorganisms, and stimulates neomicroangiogenesis. In addition, hyperbaric oxygen augments the action of aminoglycoside antibiotics. Adjuvant hyperbaric oxygen therapy should be considered in advanced or recurrent cases of malignant external otitis.
Maxillary resection defects have traditionally been technically difficult to treat with conventional radiation therapy. The irregular contours of the defect lead to an uneven distribution of the radiation doses. The potential to undertreat defects of microscopic involvement may leave residual disease. Tissue morbidity is high at isolated "hot spots" or at locations of excessive dose distribution. The use of a water-filled balloon bolus in conjunction with a balloon-retaining stent can significantly improve treatment efficacy and decrease tissue morbidity. This article describes a technique for the fabrication of a balloon-retaining stent for an orocutaneous defect.
Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.
Physiologic stimuli for differentiation of bone marrow megakaryoblasts, a critical event leading to formation of platelets, have not been identified. Therefore, we examined the effects of two potent platelet agonists, thrombin and the thromboxane A2 mimetic U46619 [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid], on the developmental regulation of cultured CHRF-288-11 megakaryoblast cells. Both thrombin and U46619 increased intracellular free calcium in fura-2 loaded, serum-deprived CHRF-288-11 cells with EC50 values of 0.07 +/- 0.01 U/ml and 194 +/- 27 nM, respectively (n = 4 each). Thrombin, but not U46619, increased intracellular pH from 7.44 +/- 0.03 to 7.55 +/- 0.06 (n = 4, P = .012) in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells. Because increases in cytosolic free calcium and/or cytosolic pH are associated with mitogenic responses in many cell systems, we determined if thrombin or U46619 stimulated CHRF-288-11 proliferation or differentiation. U46619 (1 microM) inhibited cell growth but had no effect on cell size or morphology. In contrast, thrombin (0.2 U/ml) inhibited cell division and increased overall cell size to 116 +/- 2% of control (P less than .05). Microscopically, thrombin-treated cells had increased cytoplasmic content, nuclear lobulation and nucleolar content--hallmarks of megakaryocytic differentiation. Thrombin, but not U46619, increased levels of CHRF-288-11 c-fos and c-myc mRNA transcripts (protooncogene expression). We conclude that thrombin, but not thromboxane A2, induces terminal megakaryocytic differentiation of CHRF-288-11 cells. This suggests that thrombin may act as a physiologic regulator of platelet formation in vivo.
Blood pressure (BP) may increase in hemodialysis patients during treatment of anemia with recombinant human erythropoietin (r-HuEPO). Since fluid volume is a determinant of BP in dialysis patients, changes in body fluid spaces during r-HuEPO therapy could affect BP. Thus, 51Cr-labeled red blood cell (RBC) volume, inulin extracellular fluid (ECF) volume, and urea total body water (TBW), as well as cardiac output, plasma renin activity (PRA), and plasma aldosterone concentration were determined postdialysis before and after r-HuEPO therapy in patients in whom changes in BP could be managed by ultrafiltration alone. Eleven patients entered the study: one had a renal transplant and two required addition of antihypertensive drug therapy and were excluded; eight, of whom two required antihypertensive drug therapy following the study, were included in the analyses. Results revealed an increase in predialysis hemoglobin from 67 to 113 g/L (6.7 to 11.3 g/dL) (P = 0.001) during 18 +/- 6 weeks of therapy. Predialysis diastolic BP increased from 80 to 85 mm Hg (P = 0.07), while postdialysis diastolic BP was unchanged at 73 mm Hg. 51Cr-RBC volume increased, from 0.7 to 1.3 L (P = 0.004). ECF tended to decrease, from 13.7 to 10.8 L (P = 0.064), while TBW decreased to a similar extent, but not significantly, 34.3 to 31.2 L (P = 0.16). Postdialysis ECF volume was positively correlated with mean arterial BP at baseline (r = 0.89, P = 0.007) and after therapy (r = 0.74, P = 0.035). However, the regression lines for this relationship were different (P = 0.022) before and after therapy.(ABSTRACT TRUNCATED AT 250 WORDS)