Search PubMed⌕ Search

Biomedical subjects

K J Johnson

Publications and source records attributed to K J Johnson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effect of rate and inspiratory flow on ventilator-induced lung injury.

BACKGROUND: We examined the effects of decreasing respiratory rate (RR) at variable inspiratory times (It) and reducing inspiratory flow on the development of ventilator-induced lung injury. METHODS: Forty sheep weighing 24.6+/-3.2 kg were ventilated for 6 hours with one of five strategies (FIO2 = 1.0, positive end-expiratory pressure = 5 cm H2O): (1) pressure-controlled ventilation (PCV), RR = 15 breaths/min, peak inspiratory pressure (PIP) = 25 cm H2O, n = 8; (2) PCV, RR = 15 breaths/min, PIP = 50 cm H2O, n = 8; (3) PCV, RR = 5 breaths/min, PIP = 50 cm H2O, It = 6 seconds, n = 8; (4) PCV, RR = 5 breaths/min, PIP = 50 cm H2O, It = 2 seconds, n = 8; and (5) limited inspiratory flow volume-controlled ventilation, RR = 5 breaths/min, pressure-limit = 50 cm H2O, flow = 15 L/min, n = 8. RESULTS: Decreasing RR at conventional flows did not reduce injury. However, limiting inspiratory flow rate (LIFR) maintained compliance and resulted in lower Qs/Qt (HiPIP = 38+/-18%, LIFR = 19+/-6%, p < 0.001), reduced histologic injury (HiPIP = 14+/-0.9, LIFR = 2.2+/-0.9, p < 0.05), decreased intra-alveolar neutrophils (HiPIP = 90+/-49, LIFR = 7.6+/-3.8,p = 0.001), and reduced wet-dry lung weight (HiPIP = 87.3+/-8.5%, LIFR = 40.8+/-17.4%,p < 0.001). CONCLUSIONS: High-pressure ventilation for 6 hours using conventional flow patterns produces severe lung injury, irrespective of RR or It. Reduction of inspiratory flow at similar PIP provides pulmonary protection.

Animals↗
PubMed2000

Prevention of murine influenza A virus pneumonitis by surfactant nano-emulsions.

Non-ionic surfactant nano-emulsions have extensive anti-microbial activity and are biocompatible with skin and mucous membranes at effective concentrations. Two nano-emulsion formulations (8N8 and 20N10) made from soybean oil, tributyl phosphate and Triton X-100, were tested for their ability to prevent murine influenza virus pneumonia in vivo. In the initial study, CD-1 mice were administered various dilutions of the nano-emulsions intranasally, and safe dosages and concentrations were determined. Non-toxic concentrations of the nano-emulsions were then mixed with influenza virus and applied to the nares of mice. Animals receiving mixtures of two different emulsions (8N8 or 20N10) and a LD50 of virus survived the challenge without evidence of viral infection. To determine if the nano-emulsions could prevent influenza virus infection in vivo when used as a prophylactic treatment, the nano-emulsions (8N8 at 1.0% and 20N10 at 1.0% or 0.2%) were applied to mouse nares 90 min before exposure to 5x10(5) p.f.u./ml virus by nebulized aerosol. Animals pretreated with the nano-emulsions had significantly decreased clinical signs of infection. Only 26.0% (8N8 at 1.0%), 31.25% (20N10 at 1.0%) and 37.0% (20N10 at 0.2%) of animals pretreated with nano-emulsion died from pneumonitis, whereas >80.0% of mock pretreated animals succumbed to infection (P<0.005). These findings suggest that non-ionic surfactant nano-emulsions have therapeutic potential for the prevention of influenza virus infection in vivo.

Animals↗
PubMed2000

Multiple cadherin superfamily members with unique expression profiles are produced in rat testis.

Adhesion between germ and Sertoli cells is thought to be crucial for spermatogenesis. Cadherin superfamily proteins, including classic cadherins and protocadherins, are important mediators of cell-cell adhesion. Using a degenerate PCR cloning strategy, we surveyed the expression of cadherin superfamily members in rat testis. Similar to brain, testis expressed a large number of cadherin superfamily members: 7 classic cadherins of both types I and II, 14 protocadherins, 2 protocadherin-related cadherins, and 1 cadherin-related receptorlike protein. All three protocadherin families (alpha, beta, and gamma) were found in testis. Using a semiquantitative RT-PCR assay, messenger RNA expression was determined for each cadherin superfamily member during a postnatal developmental time-course and following ablation of specific testis cell types by ethanedimethanesulfonate, methoxyacetic acid, and 2,5-hexanedione. Diverse expression patterns were observed among the cadherins, suggesting that cadherin expression is cell type-specific in testis. The large number and variety of cadherin superfamily members found in testis supports a critical function for cadherin-mediated cell-cell adhesion in spermatogenesis.

Aging↗
PubMed2000

Phlebotomy overdraw in the neonatal intensive care nursery.

OBJECTIVE: Because blood loss attributable to laboratory testing is the primary cause of anemia among preterm infants during the first weeks of life, we quantified blood lost attributable to phlebotomy overdraw, ie, excess that might be avoided. We hypothesized that phlebotomy overdraw in excess of that requested by the hospital laboratory was a common occurrence, that clinical factors associated with excessive phlebotomy loss would be identified, and that some of these factors are potentially correctable. DESIGN, OUTCOME MEASURES, AND ANALYSIS: Blood samples drawn for clinical purposes from neonates cared for in our 2 neonatal special care units were weighed, and selected clinical data were recorded. The latter included the test performed; the blood collection container used; the infant's location (ie, neonatal intensive care unit [NICU] and intermediate intensive care unit); the infant's weight at sampling; and the phlebotomist's level of experience, work shift, and clinical role. Data were analyzed by univariate and multivariate procedures. Phlebotomists included laboratory technicians stationed in the neonatal satellite laboratory, phlebotomists assigned to the hospital's central laboratory, and neonatal staff nurses. Phlebotomists were considered experienced if they had worked in the nursery setting for >1 year. Blood was sampled from a venous or arterial catheter or by capillary stick from a finger or heel. Blood collection containers were classified as tubes with marked fill-lines imprinted on the outside wall, tubes without fill-lines, and syringes. Infants were classified by weight into 3 groups: <1 kg, 1 to 2 kg, and >2 kg. The volume of blood removed was calculated by subtracting the weight of the empty collection container from that of the container filled with blood and dividing by the specific gravity of blood, ie, 1.050 g/mL. The volume of blood withdrawn for individual laboratory tests was expressed as a percentage of the volume requested by the hospital laboratory. RESULTS: The mean (+/- standard error of the mean) volume of blood drawn for the 578 tests drawn exceeded that requested by the hospital laboratory by 19.0% +/- 1.8% per test. The clinical factors identified as being significantly associated with greater phlebotomy overdraw in the multiple regression model included: 1) collection in blood containers without fill-lines; 2) lighter weight infants; and 3) critically ill infants being cared for in the NICU. Because the overall R(2) of the multiple regression for these 3 clinical factors was only.24, the random factor of individual phlebotomist was added to the model. This model showed that there was a significant variation in blood overdraw among individual phlebotomists, and as a result, the overall R(2) increased to.52. An additional subset analysis involving 2 of the 3 groups of blood drawers (ie, hospital and neonatal laboratory phlebotomists) examining the effect of work shift, demonstrated that there was significantly greater overdraw for blood samples obtained during the evening shift, compared with the day shift when drawn using unmarked tubes for the group of heavier infants cared for in the NICU. CONCLUSION: Significant volumes of blood loss are attributable to overdraw for laboratory testing. This occurrence likely exacerbates the anemia of prematurity and may increase the need for transfusions in some infants. Attempts should be made to correct the factors involved. Common sense suggests that blood samples drawn in tubes with fill-lines marked on the outside would more closely approximate the volumes requested than those without. Conversely, the use of unmarked tubes could lead to phlebotomy overdraw because phlebotomists may overcompensate to avoid having to redraw the sample because of an insufficient volume for analysis. We were surprised to observe that the lightest and most critically ill infants experienced the greatest blood overdraw. (ABSTRACT TRUNCATED)

Anemia↗
PubMed2000

Clinical performance of an in-line point-of-care monitor in neonates.

OBJECTIVE: To evaluate the bias, precision, and blood loss characteristics of an ex vivo in-line point-of-care testing blood gas and electrolyte monitor designed for use in critically ill newborn infants. STUDY DESIGN: Study participants included consecutive neonates with an umbilical artery catheter (UAC) in use for clinical laboratory testing. The in-line monitor (VIA LVM Blood Gas and Chemistry Monitoring System, VIA Medical, San Diego, CA) was directly connected to the participant's UAC and the monitor's determinations of pH, PCO(2), PO(2), sodium, potassium, and hematocrit (Hct) were compared with those simultaneously drawn and measured with a standard bench top laboratory instrument (Radiometer 625 ABL; Radiometer America, Inc, Westlake, OH). The bias (the mean difference from the reference method) and precision (1 standard deviation of the mean difference) performance criteria of the in-line monitor were derived using standard laboratory procedures. RESULTS: Sixteen neonates monitored for a total of 37 days had a total of 229 paired blood samples available for comparison by the 2 methods. Bias and precision performance characteristics of the in-line monitor were similar to reports of other point-of-care devices (ie, pH: -.003 +/-.024; PCO(2):.35 +/- 2.84 mm Hg; PO(2):.39 +/- 7.30 mm Hg; sodium:.52 +/- 2.34 mmol/L; potassium:.17 +/-.18 mmol/L; and Hct:.61 +/- 2.80%). The range of values observed for each parameter included much of the range anticipated among critically ill neonates (ie, pH: 7.15-7.65; PCO(2): 25-75 mm Hg; PO(2): 25-275 mm Hg; sodium: 127-150 mmol/L; potassium: 2.6-5.5 mmol/L; and Hct: 32%-60%). Mean blood loss (+/- standard deviation) per sample with the in-line monitor was approximately one-tenth that of the reference method: 24 +/- 7 microL versus 250 microL, respectively. There was no evidence of hemolysis and no patient related safety issues were identified with use of the in-line monitor. CONCLUSIONS: Repeated laboratory testing of critically ill neonates using an ex vivo in-line monitor designed for use in neonates provides reliable laboratory results. The blood loss and hemolysis data obtained suggests that this monitoring device offers potential for reducing neonatal blood loss-and possibly transfusion needs-during the first weeks of life. Before this promising technology can be routinely recommended for care of critically ill neonates, greater practical experience in a variety of clinical settings is needed.

Blood Gas Analysis↗
PubMed2000

Neonatal laboratory blood sampling: comparison of results from arterial catheters with those from an automated capillary device.

PURPOSE: To compare neonatal laboratory results from capillary blood samples drawn using the Tenderfoot automated capillary sampling device with those drawn through arterial catheters. DESIGN: Prospective, paired comparisons of laboratory results from capillary and arterial blood. SAMPLE: Twenty-one infants being cared for in an NICU and having indwelling arterial catheters through which a variety of predominantly glucose-containing fluids were being administered. MAIN OUTCOME VARIABLES: Statistical comparisons of paired capillary and arterial results of pH, PO2, PCO2, lactate, sodium, potassium, ionized calcium, and hematocrit. RESULTS: No capillary-arterial differences were observed for pH, PCO2, lactate, or sodium. Although capillary results were slightly, but significantly (p < .01), higher for potassium (+0.4 mEq/liter), ionized calcium (+0.47 mg/dl), and hematocrit (+4 percent), these differences fell within acceptable Clinical Laboratories Improvement Act (CLIA) performance criteria. Markedly lower PO2 (-30.2 mmHg) and glucose (-61 mg/dl) values were observed with capillary sampling. With the exception of results for PaO2 and plasma glucose, capillary blood drawn using the Tenderfoot automated device yields laboratory results comparable to those from blood drawn from arterial catheters as assessed by CLIA performance criteria.

Arteries↗
PubMed1999

Genetic linkage and radiation hybrid mapping of the three human GABA(C) receptor rho subunit genes: GABRR1, GABRR2 and GABRR3.

GABA(C) receptors mediate rapid inhibitory neurotransmission in retina. We have mapped, in detail, the human genes which encode the three polypeptides that comprise this receptor: rho1 (GABRR1), rho2 (GABRR2) and rho3 (GABRR3). We show that GABRR1 and GABRR2 are located close together, in a region of chromosome 6q that contains loci for inherited disorders of the eye, but that GABRR3 maps to chromosome 3q11-q13.3. Our mapping data suggest that the rho polypeptide genes, which are thought to share a common ancestor with GABA(A) receptor subunit genes, diverged at an early stage in the evolution of this gene family.

Amino Acid Sequence↗
PubMed1999

The compact conformation of fibronectin is determined by intramolecular ionic interactions.

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

Animals↗
PubMed1999

Cloning and chromosomal localization of human Cdc42-binding protein kinase beta.

The p21 GTPases, Rho and Cdc42, regulate numerous cellular functions by binding to members of a serine/threonine protein kinase subfamily. These functions include the remodeling of the cell cytoskeleton that is a feature of cell growth and differentiation. Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been recently characterized in rat. Both of these proteins phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility. Here we report the cDNA cloning of the human homologue of MRCKbeta, CDC42BPB, which was found by Northern blot analysis to be expressed in a wide range of tissues. The human CDC42BPB gene maps to cytogenetic band 14q32.3 by FISH analysis.

Amino Acid Sequence↗
PubMed1999

Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury.

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.

Acute Disease↗
PubMed1999

Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy.

The pathogenic mechanisms underlying myotonic dystrophy (DM), which results from a (CTG) n repeat expansion mutation in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase gene ( DMPK ), remain obscure. The multisystemic nature and variable expressivity of the symptoms are unlikely to be explained by a defect in this gene alone. However, the location of the DM-associated (CTG) n repeat in the promoter region of SIX5, immediately downstream of DMPK, implicates it as a second candidate with a pathological role in DM. We hypothesize that dysfunction of SIX5, which is homologous to the Drosophila eye development gene sine oculis ( so ), is primarily responsible for the ophthalmic features of DM. We report an expression pattern for SIX5 in the normal adult eye that matches the sites of the ocular pathology in DM. SIX5 transcripts were detected in the adult corneal epithelium and endothelium, lens epithelium, ciliary body epithelia, cellular layers of the retina and the sclera. SIX5 expression was not detected in fetal eyes. We also report a restricted but partially overlapping expression pattern for DMPK transcripts and DMPK protein in normal fetal and adult eyes. DMPK transcripts were detected in fetal eyes and in adult conjunctival and corneal epithelia, uvea, cellular layers of the retina, optic nerve and in the sclera. DMPK protein was detected in the adult retina, conjunctival and ciliary body epithelia and in the smooth muscle of the ciliary body, pupillary sphincter and uveal blood vessels. We propose that the expression patterns of these two genes indicate their relative contribution to the ophthalmological dysfunction seen in DM. Furthermore, the expression of SIX5 and not DMPK in the adult lens implicates a role for SIX5 dysfunction in the development of adult onset cataracts, the most frequently occurring eye phenotype in DM.

Adult↗
PubMed1999

The role of tumor necrosis factor-alpha in the pathogenesis of aspiration pneumonitis in rats.

BACKGROUND: Aspiration pneumonitis is characterized by proteinaceous pulmonary edema and acute infiltration of neutrophils into the alveolar space. This study examined the role of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the pathogenesis of the injury produced by the different components that may be present in the aspirate, acid, or gastric particles. METHODS: Rats were injured by intratracheal instillation of a vehicle containing acid or gastric particles. TNF-alpha concentration of bronchoalveolar lavage fluid was determined using a bioassay. upregulation of lung TNF-alpha mRNA was also measured. The effect of intratracheal anti-rat TNF-alpha treatment was assessed by lung protein permeability, blood gases, and lung myeloperoxidase activity. RESULTS: Injury vehicle alone and acid injury resulted in a small TNF-alpha peak 1-2 h after injury in the lavage fluid. Both particulate and acidic particulate groups produced a much more robust TNF-alpha signal that reached a plateau at 2-4 h after injury and declined at 8 h. Upregulation of TNF-alpha mRNA was only detected in the particulate-containing groups. Acidic particulate exposure yielded a synergistic increase in protein permeability and decrease in blood oxygenation. Anti-TNF-alpha treatment reduced protein permeability and myeloperoxidase activity and increased blood oxygenation in the groups exposed to only acid. Such treatment had no effect on either of the particulate containing injuries. CONCLUSIONS: TNF-alpha is differentially manifested according to the components that make up the aspirate but the levels of TNF-alpha expression do not correlate with the severity of the resultant injury. However, the reduction in acid-induced lung injury by anti-TNF-alpha treatment indicates that TNF-alpha plays a role in the pathogenesis of aspiration pneumonitis.

Animals↗
PubMed1999

High-dose nitric oxide inhalation increases lung injury after gastric aspiration.

BACKGROUND: Inhaled nitric oxide is often used in patients with adult respiratory distress syndrome. However, nitric oxide also may be significantly toxic, especially if administered concurrently with hyperoxia. The authors evaluated the isolated effect of nitric oxide and the combined effects of nitric oxide and hyperoxia on lung injury in rats after acid aspiration. METHODS: Animals were injured by instillation of 1.2 ml/kg hydrogen chloride in low-pH saline (the acid group) or acidified gastric particles (the casp group) into the lungs under halothane anesthesia via a tracheal catheter. Controls received no injury vehicle but rather underwent the surgical process. After recovery from anesthesia, the animals were exposed to 20% or 90% oxygen with or without 20, 40, or 80 ppm nitric oxide for 5 h. The permeability index, alveolar-arterial oxygen difference, the ratio of oxygen pressure to the inspired fraction of oxygen, and the ratio of wet to dry weight were assessed 5 h after injury as indices of lung injury. Data were assessed using analysis of variance. RESULTS: Each group included 6-10 rats. Exposure to nitric oxide (80 ppm) in air increased protein permeability in the lungs to a permeability index of 1.42+/-0.12 after acid aspiration. The combination of nitric oxide (80 ppm) and hyperoxia further increased protein leakage to a permeability index of 2.1+/-0.25. Exposure to lower concentrations of nitric oxide (e.g., 20 and 40 ppm) increased the permeability index of the lungs (1.44+/-0.21, 1.75+/-0.29, respectively) in the presence of hyperoxia, although it did not affect the permeability index of the lungs during exposure to air. Pretreatment of animals with deferoxamine and methylene blue partially inhibited the adverse effect of hyperoxia and nitric oxide, which suggested a complex underlying mechanism involving both reactive-species generation and pulmonary vasomotor changes. CONCLUSIONS: These results show that inhaled nitric oxide at 80 ppm for a short duration (5 h) increases the severity of the inflammatory microvascular lung injury after acid aspiration. The pulmonary damage is exacerbated further in the presence of high oxygen concentrations. Although lower concentrations of nitric oxide did not increase the extent of lung injury, longer exposure times need to be assessed.

Administration, Inhalation↗
PubMed1999

Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.

Animals↗
PubMed1999

Characterization of matrix metalloproteinases produced by rat alveolar macrophages.

Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145-1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, beta-casein, and kappa-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MMPs were assessed by Northern blot. Rat alveolar macrophages were found to produce a low constitutive level of MMP-2 (72-kD gelatinase A) that was only modestly upregulated following stimulation with phorbol myristate acetate, bacterial lipopolysaccharide, or immunoglobulin A-containing immune complexes. Although control cells were found to produce little or no MMP-9 (92-kD gelatinase B) or MMP-12 (metalloelastase), both enzymes were markedly upregulated upon stimulation. In the same stimulated macrophages there was little activity against type I collagen (associated with MMP-13 [collagenase-3] on the basis of Western blotting), no activity suggestive of stromelysin or matrilysin, and no measurable secretion of the serine proteinases, elastase and cathepsin G. These data demonstrate the ability of rat alveolar macrophages to elaborate certain MMPs under proinflammatory conditions, consistent with their possible involvement in the progression of acute inflammation.

Animals↗
PubMed1999

Characteristics of nonmalignant and malignant human prostate in organ culture.

Prostate tissue was obtained from 52 radical prostatectomies immediately upon surgery. From each specimen, a small piece of tissue was fixed in 10% buffered formalin and used for histology, cytokeratin staining, staining with the antibodies to the proliferation-associated antigen (Ki-67), and histochemical evaluation of the epithelial-stromal basement membrane. A second piece was used for the isolation of epithelial cells and stromal cells in monolayer culture. The remainder of each specimen was cut into cubes (approximately 1 mm on a side) and incubated in organ culture for up to 20 days. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined as described above with zero-time tissue. These studies showed that normal epithelial and stromal elements survived in organ culture in the presence of a serum-free medium containing a mixture of growth factors (epidermal growth factor, insulin, pituitary extract, and dihydrotestosterone). In many of the tissues examined at 4 days, individual glands resembled those seen immediately after surgery, with a single layer of basal epithelial cells and a layer of secretory cells above. By Day 8, the secretory epithelium was lost in many places and basal cells proliferated to fill in the lumens of the glands. All of the nonmalignant glands were reactive with the anti-cytokeratin antibody (K903), and there was a large increase in the number of cells staining for Ki-67 as compared with zero-time tissue. Staining with the Periodic Acid Schiff (PAS) and PAS-methenamine silver (PASME) reagents revealed an intact basement membrane around virtually all of the epithelial structures. The basement membrane appeared to be thickened in some areas. In places where a gland was cut during the processing of the tissue, epithelial cells migrated out of the gland and covered the cut surface of the tissue piece. There was no detectable basement membrane separating the epithelium from the stroma at these sites. Whereas nonmalignant epithelial cells were preserved in the growth factor- and dihydrotestosterone-supplemented culture medium, most of the malignant cells rapidly lysed under the same conditions. However, when phorbol myristate acetate was included in the culture medium, many of the tumor cells remained viable. This was seen with the more well-differentiated tumors as well as with tumors that were highly anaplastic. All of the tumor cells were nonreactive with anti-cytokeratin antibody, and only a few cells stained for Ki-67. The basement membrane surrounding malignant cells was thin and, in places, appeared to be discontinuous. Where malignant glands were cut in the processing of the tissue, cells did not migrate out over the cut surface. In summary, this study identifies culture conditions for the successful maintenance of human prostate tissue for several days in organ culture. Histological/histochemical features that distinguish nonmalignant and malignant tissue are present in this model.

Cell Division↗
PubMed1998

Interactions of p62(dok) with p210(bcr-abl) and Bcr-Abl-associated proteins.

A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is tyrosine-phosphorylated under a variety of circumstances including growth factor stimulation and in cells transformed by activated tyrosine kinases. A cDNA for p62(dok), reported to be the RasGAP-associated 62-kDa protein, was recently cloned from Abl-transformed cells. In this study, the interactions of p62(dok) with Bcr-Abl and associated proteins were examined. In 32D myeloid cells and Rat-1 fibroblasts transformed by p210(bcr-abl), p62(dok) is tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein. Tyrosine-phosphorylated p62(dok) from cells expressing p210(bcr-abl) bound directly to the SH2 domains of Abl and CrkL in a gel overlay assay. Previous work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in transforming fibroblasts but remains capable of inducing myeloid growth factor independence. In both fibroblasts and myeloid cells expressing this mutant, p62(dok) is underphosphorylated as compared with cells expressing full-length p210(bcr-abl) but remains capable of associating with Bcr-Abl. However, in a gel overlay assay, p62(dok) from cells expressing the SH2 domain deletion was incapable of associating directly with SH2 domains of Abl and CrkL. Interestingly, no direct binding between Bcr-Abl and p62(dok) could be demonstrated in a yeast two-hybrid assay. These data suggest that indirect interactions mediate the interaction between Bcr-Abl and p62(dok) and that the SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62(dok). Further, hyperphosphorylation of p62(dok) correlates with the ability of Bcr-Abl to transform fibroblasts but not with the induction of growth factor independence in myeloid cells.

Animals↗