Biomedical subjects

K L Nelson

Publications and source records attributed to K L Nelson.

At least 19 recordsLinked to original sources

PubMed2003

Concentrations and inactivation of Ascaris eggs and pathogen indicator organisms in wastewater stabilization pond sludge.

During treatment in wastewater stabilization ponds (WSPs) many pathogens, in particular helminth eggs, are concentrated in the sludge layer. Because periodic removal of the sludge is often required, information is needed on the concentrations and inactivation of pathogens in the sludge layer to evaluate the public health risk they pose upon removal of the sludge. In this paper, previous reports on the sludge concentrations of various pathogen indicator organisms and helminth eggs are reviewed and results from our own recent experiments are reported. The advantages and disadvantages of several methods for studying inactivation in the sludge layer are discussed, as well as implications for the management of WSP sludge. In our recent experiments, which were conducted at three WSPs in central Mexico, sludge cores, dialysis chambers, and batch experiments were used to measure the inactivation rates of fecal coliform bacteria, fecal enterococci, F+ coliphage, somatic coliphage, and Ascaris eggs. The first-order inactivation rate constants were found to be approximately 0.1, 0.1, 0.01, 0.001, and 0.001 d(-1), respectively. The concentrations of all the organisms were found to vary both vertically and horizontally in the sludge layer; therefore, to determine the maximum and average concentration of organisms in the sludge layer of a WSP, complete sludge cores must be collected from representative locations throughout the pond.

Animals↗

PubMed2001

Inactivation of viable Ascaris eggs by reagents during enumeration.

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.

Animals↗

PubMed2001

Chyloabdomen in a mature cat.

A mature, castrated male cat presented with progressive lethargy and a severely distended abdomen. Abdominal radiographs, abdominocentesis, and evaluation of the fluid obtained led to a diagnosis of chyloabdomen. The underlying pathology, etiology, diagnosis, and treatment associated with this disease are discussed.

Abdominal Pain↗

PubMed2000

Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts.

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.

Alkaline Phosphatase↗

PubMed2000

Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Anemia, Aplastic↗

PubMed1999

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

Animals↗

PubMed1999

Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Bacterial Toxins↗

PubMed1999

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein.

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Aeromonas↗

PubMed1999

Channels formed by subnanomolar concentrations of the toxin aerolysin trigger apoptosis of T lymphomas.

Aerolysin is a channel-forming toxin that binds to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on target cells. Here, we show that subnanomolar concentrations of aerolysin trigger apoptosis of T lymphomas. Using inactive aerolysin variants, we determined that apoptosis was not directly triggered by binding to GPI-anchored receptors, nor was it caused by receptor clustering induced by toxin oligomerization. Apoptosis was caused by the production of a small number of channels in the cell membrane. Channel formation resulted in a rapid increase in intracellular calcium, which may have been the signal for apoptosis. Overexpression of the antiapoptotic protein bcl-2 blocked aerolysin-induced apoptosis, although this effect was overcome at higher toxin concentrations.

Animals↗

PubMed1998

Activation of STAT3 by the c-Fes protein-tyrosine kinase.

STATs (signal transducers and activators of transcription) are transcription factors that contain SH2 domains and are activated by tyrosine phosphorylation, often in response to cytokine stimulation. Recent evidence indicates that the transforming tyrosine kinases encoded by the v-Src, v-Abl, and v-Fps oncogenes can induce STAT activation, suggesting that their normal cellular homologs may contribute to STAT activation under physiological conditions. In this report, we provide direct evidence that c-Fes, the normal human homolog of v-Fps, potently activates STAT3. Transient transfection of human 293T cells with STAT3 and Fes resulted in strong stimulation of STAT3 DNA binding activity. In contrast, only modest activation of STAT5 by Fes was observed in this system, indicative of possible selectivity. To determine whether Fes-induced STAT3 activation is dependent upon endogenous mammalian kinases, co-expression studies were also performed in Sf-9 insect cells. Fes also induced a dramatic increase in STAT3 DNA binding activity in this system, whereas no activation of STAT5 was observed. As a positive control, both STAT3 and STAT5 were shown to be activated by the Bcr-Abl tyrosine kinase in Sf-9 cells. Fes induced strong tyrosine phosphorylation of STAT3 in both expression systems, consistent with the gel-shift results. Fes and STAT3 have been independently linked to myeloid differentiation. Results presented here suggest that these proteins may cooperate to promote differentiation signaling in response to hematopoietic cytokines.

Animals↗

PubMed1998

Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin.

Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin of Trypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.

Animals↗

PubMed1998

Movement of a loop in domain 3 of aerolysin is required for channel formation.

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

Aeromonas↗

PubMed1997

The glycosylphosphatidylinositol-anchored surface glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin.

Aerolysin is a channel-forming protein secreted by virulent Aeromonas spp. Some eucaryotic cells, including T-lymphocytes, are sensitive to very low concentrations of the toxin (<10(-9) M). Here we show that aerolysin binds selectively and with high affinity to the glycosylphosphatidylinositol (GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte populations as well as in brain. Less than 1 ng of purified Thy-1 could be detected by probing Western blots with the toxin. Mutant T-cell lines that lack the ability to add GPI anchors to Thy-1 and other surface proteins were much less sensitive to aerolysin, as were wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins. Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in their membranes were much more sensitive to aerolysin than protein-free liposomes.

Animals↗

PubMed1997

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein.

The plasma membrane of rat erythrocytes contains a 47-kDa glycoprotein that binds the channel-forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N-terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N-termini of a group of membrane proteins that are thought to be involved in ADP-ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C-terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol-specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells' sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin-binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.

Animals↗

PubMed1997

Constitutive activation of Stat3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells.

Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive mutant of v-Src, we determined that Stat3 is activated within 15 min of shift from nonpermissive to permissive temperature for cell transformation. This finding indicates that v-Src tyrosine kinase activity is required for Stat3 activation and suggests that Stat3 is proximal to signaling initiated by Src. In addition, Stat3 activation is induced by another nonreceptor tyrosine kinase, v-Fps; by polyoma virus middle T antigen, which activates Src family kinases; and by v-Sis, which acts as a ligand for the platelet-derived growth factor receptor. In contrast SV40 large T antigen, which transforms cells through different mechanisms, and the v-Ras and v-Raf oncoproteins, which lie in signaling pathways downstream of tyrosine kinases, do not activate Stat3. We did not detect significant activation of Stat1, Stat5, or Stat6 in fibroblasts transformed by the viral oncoproteins investigated. Moreover, Stat3 is activated in response to epidermal growth factor (EGF) but not heregulins in immortalized normal human breast epithelial cells. Because constitutive activation of c-Src and EGF receptor kinases is associated with the progression of breast cancer, we examined activation of STATs in human cell lines derived from breast carcinomas. We detected constitutive activation of Stat3 in five of nine breast carcinoma cell lines but not in normal breast epithelial cells. Furthermore, experiments with an EGF receptor-specific inhibitor indicated that the constitutive activation of Stat3 in these breast carcinoma cell lines is not necessarily dependent on signaling through the EGF receptor, although EGF stimulation further increases Stat3 activation. Taken together, our results demonstrate that selective activation of Stat3 is a common event during oncogenic transformation that directly or indirectly involves activation of specific tyrosine kinase signaling pathways.

3T3 Cells↗

PubMed1996

Potential for H-DNA in the human MUC1 mucin gene promoter.

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.

Base Sequence↗

PubMed1995

Clinical safety of gadopentetate dimeglumine.

PURPOSE: To quantify the rate of adverse reactions to gadopentetate dimeglumine. MATERIALS AND METHODS: Magnetic resonance (MR) imaging was performed in 15,496 patients in April-September 1992. Data were collected before and after intravenous administration of 0.1 mmol/kg gadopentetate dimeglumine. RESULTS: Adverse reactions occurred in 2.4% (n = 372) of patients. Symptoms abated the same or next day in 94.1% (n = 350). Whereas onset occurred within 30 minutes after injection in 49.7% (n = 185), onset occurred more than 1 hour after injection in 44.9% (n = 167). Two serious adverse reactions occurred and were attributed to underlying disease. The rate of adverse reaction was 3.7% in patients with a history of asthma (31 of 831 patients) or allergy (144 of 3,860 patients). Patients with previous reactions to an MR imaging or iodinated contrast agent had an adverse-reaction rate in this study of 21.3% (16 of 75) and 6.3% (54 of 857), respectively. The rate of adverse reaction was 2.2% when gadopentetate dimeglumine was administered slowly and 2.9% when it was administered rapidly. CONCLUSION: Findings confirm the safety of gadopentetate dimeglumine.

Asthma↗

PubMed1995

Basic principles of MR contrast.

The use of intravenous contrast media is well established in magnetic resonance (MR) for improved diagnosis. MR differs from other imaging modalities in the complexity of signal and contrast dependence, with the method of measurement having great impact on tissue contrast. Unlike computed tomography, in which contrast depends solely on x-ray density, the signal intensity on MR is determined by differences in spin density, T1 and T2 relaxation times, diffusion, perfusion, and magnetic susceptibility. The only contrast agents in widespread clinical use at this time are paramagnetic metal ion chelates. These compounds influence tissue contrast by enhancing T1 and T2 relaxation. The effectiveness of any one agent is dependent on a number of basic parameters, including concentration, number of coordination sites, magnetic moment, distance between the ion and water protons, and correlation times. An understanding of general contrast mechanisms, principles of contrast agent design, and basic MR imaging techniques is important for proper clinical implementation and medical diagnosis.

Central Nervous System Diseases↗