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P R Johnson

Publications and source records attributed to P R Johnson.

At least 19 recordsLinked to original sources
PubMed2002

Correlation of NADH and Ca2+ signals in mouse pancreatic acinar cells.

Relationships between calcium signals and NADH responses were investigated in pancreatic acinar cells stimulated with calcium-releasing secretagogues. Cytosolic calcium signals were studied using Fura Red or calcium-sensitive Cl(-) current. Mitochondrial calcium was measured using Rhod-2. The highest levels of NADH autofluorescence were found around the secretory granule region. Stimulation of cells with physiological doses of cholecystokinin (CCK) triggered slow oscillations of NADH autofluorescence. NADH oscillations were clearly resolved in the mitochondrial clusters around secretory granules. Very fast apical calcium signals induced by acetylcholine (ACh) produced no detectable changes in NADH; slightly more extended apical (or preferentially apical) calcium transients triggered clear NADH responses. Triple combined recordings of cytosolic calcium, mitochondrial calcium and NADH revealed the sequence of development of individual signals: an increase in cytosolic calcium was accompanied by a slower mitochondrial calcium response followed by a delayed increase in NADH fluorescence. Recovery of cytosolic calcium was faster than recovery of mitochondrial calcium. NADH recovery occurred at elevated mitochondrial calcium levels. During the transient cytosolic calcium oscillations induced by intermediate doses of ACh, there was an initial increase in NADH fluorescence following the first calcium transient; each of the subsequent calcium responses produced biphasic NADH changes comprising an initial small decline followed by restoration to an elevated calcium level. During the higher-frequency sinusoidal calcium oscillations induced by higher doses of ACh, NADH responses fused into a smooth rise followed by a slow decline. Supramaximal doses of ACh and CCK produced single large NADH transients.

Acetylcholine↗
PubMed2002

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells.

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.

Acetylcholine↗
PubMed2001

Mechanisms of airway remodeling. Airway smooth muscle.

The airway smooth muscle cell can contract; relax; participate in allergic and inflammatory responses by expressing adhesion molecules, releasing cytokines, and producing matrix proteins and proteases; and, as has been reported, undergo migration. These properties enable the muscle cell to be a key component in the airway wall remodeling that accompanies persistent asthma. Evidence is emerging that identifies the pivotal steps in the signal transduction pathways that lead to the excessive proliferation of the muscle observed in vitro in airway smooth muscle cells from subjects with asthma. The contractile, biochemical, and growth characteristics of muscle from allergic subjects are different from those of nonallergic subjects. In addition, the allergic response impacts on the extracellular matrix in which the muscle is embedded, by altering the profile of matrix proteins released. Once the relationships between allergy and inflammation of the smooth muscle and its extracellular matrix are better defined, opportunities to prevent or reverse airway remodeling will become available.

Allergens↗
PubMed2001

Airway smooth muscle cell proliferation is increased in asthma.

UNLABELLED: Increased airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely to be the result of increased muscle proliferation. We have for the first time been able to culture ASM cells from asthmatic patients and to compare their proliferation rate with that of nonasthmatic patients. Asthmatic ASM cell cultures (n = 12) were established from explanted lungs and endobronchial biopsies. Nonasthmatic ASM cells (n = 10) were obtained from explanted tissue from patients with no airway disease, emphysema, carcinoma, and fibrosing alveolitis. Cell counts, tritiated thymidine incorporation, and cell cycle analysis were conducted over 7 d. Asthmatic ASM cell numbers at Days 3, 5, and 7 were significantly higher than corresponding values for nonasthmatic cells (p < 0.05). Tritiated thymidine incorporation was increased 3.2-fold in asthmatic cells compared with nonasthmatic cells within the first 24 h (p = 0.026). Flow cytometric analysis of DNA content on Days 1 and 2 revealed that a significantly greater percentage of asthmatic ASM cells were in the G2 + M phase (p < 0.05). This study shows for the first time that proliferation of ASM cells is increased in patients with asthma and provides evidence for an intrinsic abnormality in the ASM cell in this disease. KEYWORDS: asthma; human airway smooth muscle; cell culture; cell proliferation; hyperplasia

Adolescent↗
PubMed2001

Trisomy 14 in myeloid malignancies: report of two cases and review of the literature.

To our knowledge, 58 cases of trisomy 14 in association with hematological malignancies have been reported, predominantly in myeloid malignancies. We report two patients with trisomy 14 associated with myelodysplasia. The bone marrow showed trilineage dysplasia, monocytosis and only mild thrombocytopenia. A nonmosaic karyotype was seen in both patients and survival from diagnosis was short (<1 year). The features are consistent with data from other published cases and support the hypothesis that trisomy 14 is a non-random karyotypic abnormality, with defined clinical associations and a poor prognosis.

Aged↗
PubMed2001

Panniculectomy with simultaneous gynecologic oncology surgery.

OBJECTIVE: The objective was to report the utility and morbidity of panniculectomy in obese gynecologic oncology patients undergoing exploratory laparotomy. METHODS: A retrospective chart review of 41 consecutive women who had a panniculectomy as part of an abdominal gynecologic oncology procedure between July 1996 and May 2000 was performed. Obese patients possessing a large pannus, the majority with a BMI > or = 30 kg/m(2), were included. Demographic, preoperative, operative, and postoperative data were obtained. Statistical analyses were performed using Statistical Analysis System (SAS) Version 6.13. RESULTS: Panniculectomy was performed on 41 patients with a mean age of 55, weight of 126 kg, and BMI of 48 kg/m(2). The most common comorbidities in this population were hypertension, diabetes, and osteoarthritis. Wound infection occurred in 4 (9.8%) patients; 88% of the patients received a hysterectomy. The average EBL was 358 cc. Operative time and length of hospital stay were on average 203 min and 5.5 days, respectively. A prior history of diabetes increased the risk of early complications (P = 0.03). Late complications were more likely to occur in older women (P = 0.05). Wound complications were increased in patients with larger BMI's (P = 0.05). CONCLUSIONS: This study supports the safety of the panniculectomy procedure in this high-risk group of morbidly obese patients for whom a technical advantage may be achieved by improved operative exposure.

Adult↗
PubMed2001

Factors controlling transduction signaling and proliferation of airway smooth muscle.

The airway smooth muscle cell is an active participant in the inflammatory response that accompanies asthma. It can interact with the epithelium and inflammatory cells to produce cytokines and cell surface molecule upregulation. Moreover, smooth muscle cells can alter the composition of the extracellular matrix proteins via changes in the production of matrix metalloproteinases and their tissue inhibitors. These properties may contribute to the increase in the amount of airway smooth muscle that characterizes the asthmatic airway wall and the remodeling that underlies the structural changes that lead to persistent asthma.

Asthma↗
PubMed2001

Role of human airway smooth muscle in altered extracellular matrix production in asthma.

1. The underlying abnormality in asthma is not fully understood; however, inflammation, airway remodelling and bronchial hyperresponsiveness are key factors. The plasma exudate from the microvascular leakage plays a significant role in remodelling, which includes extracellular matrix (ECM) protein deposition/breakdown and airway smooth muscle (ASM) hyperplasia/hypertrophy. 2. The ECM is an intricate network of macromolecules that forms the 'scaffolding' of the airways. This scaffolding not only acts as mechanical support that plays a crucial role in the maintenance of airway function and structure, but it is also a dynamic and complex network that has the potential to influence cellular function, including migration, differentiation and proliferation of a number of cell types. 3. In asthmatic airways, the profile of ECM proteins is altered. The deposition of collagen I, III, V, fibronectin, tenascin, hyaluronan, versican and laminin alpha2/beta2 is increased, whereas the deposition of collagen IV and elastin is decreased. 4. This imbalance in the ECM profile within the asthmatic airway could be due to: (i) increased de novo synthesis of ECM proteins; (ii) decreased activity of its degrading enzymes, namely matrix metalloproteinases (MMP); or (iii) upregulation of the tissue-specific inhibitors of metalloproteinases (TIMP). 5. One of the characteristic features of asthma is an increase in the amount of ASM within the airways. The ECM proteins/MMP/TIMP in and around the smooth muscle may play a contributory role in this increased growth. 6. The role of current asthma treatments in the prevention or reversal of airway ECM changes is an area that has only recently become of interest, with the majority of the in vivo work focusing on the effects of corticosteroids. 7. The evidence presented in this review indicates that the ASM may influence its own environment/proliferation through the production of ECM proteins, MMP and TIMP. Further studies are needed to fully understand the role of the ASM in the production of ECM proteins, MMP and TIMP andtheir potential influence in the mechanisms underlying asthma.

Adult↗
PubMed2001

Tumour necrosis factor-alpha potentiates contraction of human bronchus in vitro.

OBJECTIVE: Chronic inflammation of the airways is an important component in the induction of airway hyperresponsiveness (AHR) in asthma. The pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) have been implicated in the induction of AHR. Whether these cytokines directly modulate the contractile properties of human airway smooth muscle (ASM) has not been fully investigated. METHODOLOGY: The contractile response to acetylcholine (ACh) (10(-8) to 10(-3) mol/L) was determined in isolated human bronchial segments both prior to and following a 16-h incubation period with IL-1beta (10 or 20 ng/mL) and TNF-alpha (25 ng/mL), either alone or in combination. Incubation of human bronchial segments with IL-1beta/TNF-alpha was also performed in the presence of the COX-1/COX-2 inhibitor, indomethacin. RESULTS: Tumour necrosis factor-alpha potentiated the contractile response to ACh by approximately 27%, while IL-1beta or the cytokines in combination had no effect. Indomethacin had no modulatory effect on the contractile response to ACh in the cytokine-treated tissues. CONCLUSIONS: The relative concentrations of IL-1beta/TNF-alpha in the vicinity of ASM may ultimately determine their effects on ASM contraction in asthma.

Acetylcholine↗
PubMed2001

ERK activation and mitogenesis in human airway smooth muscle cells.

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.

Blood Proteins↗
PubMed2001

Functional effects of protease-activated receptor-2 stimulation on human airway smooth muscle.

The protease-activated receptor (PAR)-2 is present on the smooth muscle and epithelium of human airways and can be activated by mast cell tryptase, trypsin, or the PAR-2 activating peptide (AP). Trypsin and the PAR-2 AP induced contractions in human isolated airways, and these contractions were potentiated in the presence of the cyclooxygenase (COX) inhibitor indomethacin. Trypsin also increased the contractions to histamine in airways from sensitized (allergic) patients but not from nonsensitized (nonallergic) patients. Tryptase purified from human lung, skin and lung recombinant beta-tryptases, trypsin, and the PAR-2 AP all increased DNA synthesis in human airway smooth muscle (HASM) cells. Activation of PAR-2 by tryptase, trypsin, and the PAR-2 AP did not induce PGE(2) release from HASM cells. Trypsin and the PAR-2 AP increased the levels of intracellular calcium in HASM cells, with desensitization evident after treatment with either agonist. In conclusion, activation of PAR-2 can induce contractions of human airways, potentiate contractions to histamine, and induce proliferation and therefore may contribute to airway diseases such as asthma.

Adult↗
PubMed2001

Gene delivery of vaccines for infectious disease.

Genetic immunization is the process of delivering and expressing a gene (or therapeutic nucleic acid) encoding a pathogen-derived antigen into target host cells to elicit a protective humoral or cell-mediated immune response. Gene delivery methods to achieve this goal have expanded rapidly, and currently employ a variety of oligonucleotides, synthetic polypeptides, recombinant vectors and even edible plants, all of which have been shown to be capable of inducing protective immunity in experimental animal models. This review highlights recent progress in several gene delivery systems (both non-viral and viral methods) using novel in vivo approaches to engender effective host immune responses against the introduced antigen.

Adenoviridae↗
PubMed2000

An epithelial-derived factor inhibits contraction in canine perfused bronchial segments.

We have previously reported, using a novel preparation of canine airway segments, that the sensitivity of acetylcholine was greater when applied to the adventitial (outside) surface than the epithelial (inside) surface. The present study investigated if this "barrier-effect" was partly the result of pharmacological modulation by the epithelium. As previously demonstrated, canine airway segments were less sensitive to inside than outside application of acetylcholine (pD(2) 3.0+/-0.4 and 4.5+/-0.4, respectively, P<0.001, n=5). The addition of donor bronchi significantly decreased the sensitivity of the airway segment to outside application of acetylcholine (pD(2) 4.3+/-0.2 and 3.6+/-0.2, respectively, P<0.002, n=4). Indomethacin (2.5 microM) treatment of both the donor bronchi and the airway segment and removal of donor epithelium abolished the rightward shift in the acetylcholine-response curves. In addition, inhibition of cyclooxygenase within the airway segments themselves, but not the donor bronchi, also inhibited the rightward shift in the curves. These results indicate that the donor epithelium is capable of pharmacologically modulating responses of the airway segment to outside applied acetylcholine by producing an epithelial-derived factor, which in turn causes the release of a downstream cyclooxygenase product from within the airway segment.

Acetylcholine↗
PubMed2000

Recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse.

The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.

Animals↗
PubMed2000

Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines.

Gene transfer vectors based on adeno-associated virus mediate high-level, stable gene expression in a variety of postmitotic tissues; thus, there is interest in developing improved production systems. We previously described the generation of rAAV producer cell lines that, upon infection with adenovirus, yielded biologically active rAAV particles. In these studies we show that the adenovirus multiplicity of infection (m.o.i.) is a critical variable for efficient production of cell line-derived rAAV and can affect yields by over 200-fold. Moreover, a threshold level of adenovirus was found necessary for high-titer vector production. To define the possible factors responsible for adenovirus m.o.i. -dependent rAAV yields, we analyzed rep and cap expression as a function of adenovirus m.o.i. High-level AAV capsid protein synthesis was observed in rAAV producer cells at adenovirus m.o.i. > or =10. This prompted us to analyze the rep-cap copy number following adenovirus infection. We documented robust episomal DNA amplification (100-fold) of integrated rep-cap sequences. Interestingly, no amplification of rep-cap sequences was observed when the sequences (in plasmid form) were transfected into adenovirus-infected HeLa cells. These data suggest that adenovirus-dependent rep-cap gene amplification is a critical process responsible for efficient rAAV synthesis in stable cell lines.

Adenoviridae↗
PubMed2000

Augmented insulinotropic action of arachidonic acid through the lipoxygenase pathway in the obese Zucker rat.

OBJECTIVE: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose-stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. RESEARCH METHODS AND PROCEDURES: Islets were isolated from 10- to 12-week-old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12-lipoxygenase metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), was measured by enzyme immunoassay. RESULTS: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (<11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA-induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12-lipoxygenase, esculetin (0.5 microM), inhibited AA-stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12-HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. DISCUSSION: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12-lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.

Animals↗
PubMed2000

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector.

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.

Anemia↗