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Biomedical subjects

E J Carlson

Publications and source records attributed to E J Carlson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.

The Sod2 gene for Mn-superoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide.

Acidosis↗
PubMed1994

The mapping of transgenes by fluorescence in situ hybridization on G-banded mouse chromosomes.

A highly sensitive method for the mapping of transgenes and other genes in the mouse genome is described. This technique combines high-resolution G-banding and fluorescence in situ hybridization (FISH) with either biotin/avidin-FITC or digoxigenin-anti-digoxigenin-FITC, the latter being the more sensitive. Banding patterns are obtained with trypsin/Giemsa-treated slides, and sensitivity is greatly increased by the use of mouse Cot-1 DNA. With this protocol, four different 14.5-kb human Cu/Zn-superoxide dismutase transgene insertions ranging in copy number from 2 to 8 have been localized to four different mouse chromosomes. The utility and sensitivity of this procedure were verified with a Chromosome (Chr) 16-specific cosmid probe, H22, as well as with the mapping of a high-copy-number human beta-amyloid/A4 transgene.

Animals↗
PubMed1992

Relationship of resistance to oxygen free radicals to CuZn-superoxide dismutase activity in transgenic, transfected, and trisomic cells.

Although CuZn-superoxide dismutase (CuZnSOD) has been shown to reduce oxidative damage in several systems, the quantitative relationship between the degree of protection and CuZnSOD activity has not been well investigated. Therefore, the ability of cells to tolerate superoxide toxicity was assessed as a function of endogenous CuZnSOD activity in several mouse and human cell lines with progressively higher levels of CuZnSOD activity. In five lines of fetal fibroblasts derived from SOD1-transgenic mice, with CuZnSOD activities of 1.7- to 7.1-fold the nontransgenic level and no changes in the cellular glutathione peroxidase (GSHPx) activity, a direct relationship (r = 0.97) between the LD50 to paraquat and enzyme activity was observed, suggesting that CuZnSOD activity is the single most important factor in determining the paraquat LD50. Mouse trisomy 16 fetal fibroblasts and human trisomy 21 lung fibroblasts, both expressing a 1.5-fold increase in CuZnSOD activity, were 1.5-fold more tolerant to paraquat than were their diploid counterparts. Furthermore, the protective effect of CuZnSOD at the DNA level, as shown by reduced thymine glycol generation, was demonstrated in paraquat-treated transgenic fibroblasts. A direct relationship (r = 0.78) of paraquat LD50 and CuZnSOD activity was also observed with a panel of six lines of SOD1- transfected HeLa cells with 1.6- to 7.3-fold the basal CuZnSOD activity. Moreover, there was no correlation between resistance to paraquat toxicity and the cellular GSHPx and/or catalase activity. Taken together, these results demonstrate a consistently protective effect of endogenous CuZnSOD against superoxide toxicity in both primary and transformed cell lines.

Animals↗
PubMed1992

The role of superoxide anions in the establishment of an interferon-alpha-mediated antiviral state.

It has been suggested that CuZn-superoxide dismutase (CuZnSOD) is required for the establishment of an interferon (IFN)-mediated antiviral state. To investigate this possibility further, a panel of 6 stably transfected HeLa clones, expressing CuZnSOD activity from 1.6 to 7.3 times the normal level, were treated with different concentrations of recombinant human interferon alpha A (rHuIFN-alpha A) followed by challenge with vesicular stomatitis virus (VSV). A biphasic response curve was generated (r = 0.87, p less than 0.025). Clones with up to 3-fold basal level CuZnSOD activity exhibited an inverse relationship between their ability to generate an IFN-alpha-mediated antiviral state and CuZnSOD activity: the higher the CuZnSOD activity, the lower the sensitivity to IFN-alpha and the more IFN-alpha required for antiviral defense. Clones with between 4 to 7.3 times higher CuZnSOD activity than the non-transfected HeLa control showed a direct relationship between the CuZnSOD activity and the sensitivity to IFN-alpha. Furthermore, in agreement with the results obtained with the SOD1-transfected HeLa cells with up to 3 times the basal SOD activity, fetal fibroblasts derived from SOD1-transgenic mouse strains, TgHS-229 and TgHS-218, which also express 3 times the basal CuZnSOD activity, required higher IFN-alpha to achieve 50% protection. These results suggest a possible role for superoxide anion in the establishment of IFN-mediated antiviral effect, especially in the dose-response region in which the inverse relationship between the generation of the IFN-alpha-mediated antiviral state and CuZnSOD activity was observed. To assess this possibility, allopurinol was used as a xanthine oxidase inhibitor and hydroxyl radical scavenger in the IFN-alpha-mediated antiviral assay. Addition of 3 mM allopurinol diminished the IFN-mediated antiviral effect by between 40 and 50% (p less than 0.01), and there was a reduction in superoxide generation (p less than 0.05). The degree of reduction caused by allopurinol treatment was higher at an IFN-alpha concentration of 10 U/ml than at 100 U/ml, and there was no correlation between CuZnSOD activity and the degree of reduction. To establish further the role of superoxide as an antiviral agent, paraquat was used as a superoxide generator in the absence of IFN-alpha in the antiviral assay. Although paraquat at high concentrations is toxic to the cells, it actually showed a protective effect against VSV infection, and an inverse relationship (r = 0.79, r less than 0.025) between cell survival and CuZnSOD activity was observed with 150 mM paraquat treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Allopurinol↗
PubMed1991

Sequence diversity of the 60-kilodalton protein and of a putative 15-kilodalton protein between the trachoma and lymphogranuloma venereum biovars of Chlamydia trachomatis.

DNA from Chlamydia trachomatis serovars L3, C, and E corresponding to the open reading frames of the 60-kDa protein and of a putative 15-kDa protein was sequenced. The open reading frames coding for the 60-kDa protein had 1,641 bp in the three serovars. Compared with the L3 serovar, there were 9 and 11 amino acid changes in the C and E serovars, respectively. The open reading frames corresponding to the putative 15-kDa protein had 450, 456, and 453 bp for the L3, C, and E serovars, respectively. When compared with the L3 serovar, the C and E serovars had 14 and 16 amino acid differences, respectively.

Amino Acid Sequence↗
PubMed1991

Clinical and pathologic correlation of endometrial cavity fluid detected by ultrasound in the postmenopausal patient.

A registry of ultrasound procedures spanning nearly 5 years was searched retrospectively to discover cases of endometrial cavity fluid collections in postmenopausal women. Twenty cases were identified; all medical records were available for review. One patient was lost to follow-up. Seventeen patients had surgical procedures: 11 had only a D&C, and six had a primary evaluation of laparotomy with removal of the uterus and adnexa. Five women had cancer (two ovarian, one tubal, one endometrial, and one cervical); eight women had benign gynecologic conditions, including uterine fibroids (five), ovarian serous cystadenoma (two), and cervical dysplasia (one). There were two cases of apparent subclinical pyometra. Five women had endometrial pathology consistent with prescribed hormone therapy for breast cancer (four) or endometrial hyperplasia (one).

Aged↗
PubMed1990

Attenuation of glutamate-induced neuronal swelling and toxicity in transgenic mice overexpressing human CuZn-superoxide dismutase.

The role of oxygen-derived free radicals, superoxide in particular, in the pathogenesis of neuronal cell death induced by glutamate was studied using primary culture cortical neurons from transgenic mice overexpressing human CuZn-superoxide dismutase. Primary cortical neuron cultures were developed form 15-day-old fetuses of both transgenic mice and their normal littermates. Both human CuZn-superoxide dismutase and host mouse CuZn-superoxide dismutase activities in cultured neurons were identified by native gel electrophoresis followed by nitroblue tetrazolium staining. Cultured neurons grown for 10-12 days in vitro were exposed briefly to 0.5 mM glutamate for 5 minutes, followed by biochemical and morphological examinations at 2 and 4 hours. Our data have demonstrated that glutamate neurotoxicity is significantly reduced in transgenic neurons at 2 and 4 hours following exposure to glutamate, as measured by the intracellular 3-0-methyl glucose space, the efflux of lactate dehydrogenase, and by phase-contrast and bright-field trypan blue staining. These data indicate that transgenic neurons containing two- to threefold the normal amount of CuZn-superoxide dismutase activity are protected against glutamate neurotoxicity in vitro. Our results suggest that oxidative stress play an important role in glutamate-induced neuronal swelling and toxicity.

3-O-Methylglucose↗
PubMed1990

Reduced neurotoxicity in transgenic mice overexpressing human copper-zinc-superoxide dismutase.

The role of oxygen-derived free radicals, superoxide in particular, in the pathogenesis of neuronal cell death induced by glutamate was studied using cultured cortical neurons from transgenic mice overexpressing human copper-zinc-superoxide dismutase. Primary cortical neuron cultures were developed from 15-day-old fetuses of both transgenic mice and their normal littermates. Both human copper-zinc-superoxide dismutase and host mouse copper-zinc-superoxide dismutase activities in cultured neurons were identified by native gel electrophoresis followed by nitroblue tetrazolium staining. Cultured neurons grown for 10-12 days in vitro were exposed briefly to 0.5 mM glutamate for 5 minutes, followed by biochemical and morphological examinations at 2, 4, and 24 hours. Our data have demonstrated that glutamate neurotoxicity is significantly reduced in transgenic neurons at 2 and 4 hours following exposure to glutamate, as measured by the efflux of lactate dehydrogenase, the 3-O-methyl glucose space, and by phase-contrast and bright-field trypan blue staining. These data indicate that transgenic neurons containing twofold to threefold the normal amount of copper-zinc-superoxide dismutase activity as the result of expression of the human copper-zinc-superoxide dismutase transgene are protected against glutamate neurotoxicity in vitro. Our results suggest that oxidative stress, at least in part, plays an important role in the biochemical pathways amplifying N-methyl-D-aspartate receptor-mediated neurotoxicity.

Animals↗
PubMed1989

Cloning and characterization of a Chlamydia trachomatis L3 DNA fragment that codes for an antigenic region of the major outer membrane protein and specifically hybridizes to the C- and C-related-complex serovars.

Chlamydia trachomatis L3 DNA was cloned and expressed in lambda gt11. A recombinant plaque that expressed an antigen that reacted with rabbit polyclonal antichlamydial L3 serum and with two monoclonal antibodies specific for serovars L3 and I was selected from this Chlamydia genomic library. The beta-galactosidase Chlamydia fusion protein was purified by immunoaffinity chromatography and injected into mice to produce monoclonal antibodies. These monoclonal antibodies reacted by Western (immuno-) blot with both the fusion protein and the major outer membrane protein from purified L3 elementary bodies. The chlamydial DNA fragment was shown by DNA sequence analysis to be 168 base pairs in length and to correspond to the constant regions 1 and 2 and the variable segment 1 of the major outer membrane protein gene. The recombinant chlamydial DNA fragment hybridized under stringent conditions by Southern and dot blot analysis exclusively with the DNA from the C- and C-related-complex C. trachomatis serovars.

Amino Acid Sequence↗
PubMed1987

Ultrastructural analysis of the anti-chlamydial activity of recombinant murine interferon-gamma.

The effect of murine interferon-gamma (MuIFN-gamma) on the developmental cycle of Chlamydia trachomatis in McCoy cells was analyzed by light and electron microscopy. Addition to the culture media of 10 ng/ml of MuIFN-gamma, either 24 hr before or immediately after Chlamydia infection, resulted in a significant inhibition of the growth of this organism. Microscopic analysis showed that with both treatments the majority of microorganisms were arrested at the elementary body stage. Only a few small chlamydial inclusions were detected at 48 hr postinfection and contained predominately reticulate bodies. Furthermore, the growth of Chlamydia was arrested in cells that were treated with MuIFN-gamma at various intervals following infection. Addition of MuIFN-gamma at 8 or 12 hr after infection resulted in the arrest of chlamydial growth before initiation of reticulate body fission. When the MuIFN-gamma was added 24 hr postinfection, we could detect, by electron microscopy, inhibition at the stage of reticulate body replication.

Animals↗
PubMed1985

Evidence that the beta-adrenergic system and prostaglandins stimulate renin release through different mechanisms.

In normal man, converting enzyme inhibition (CEI) acutely increases plasma active renin and decreases plasma inactive renin. This reciprocal relationship suggests that conversion of inactive to active renin may be important in the acute response to stimulation of renin secretion. To determine whether the beta-adrenergic system or prostaglandins (PGs) participate in the acute effect of CEI on renin, we administered captopril (50 mg) alone and with either propranolol (P; 80 mg) or a PG cyclooxygenase inhibitor [PI; indomethacin (50 mg) or ibuprofen (800 mg)] to normal subjects ingesting a 25 meq/day Na diet. Supine blood pressure fell by 12 +/- 2 (+/- SE) mm Hg with CEI alone, 10 +/- 1 mm Hg with CEI plus P, and 7 +/- 1 mm Hg with CEI plus PI. Active renin rose 8-fold (P less than 0.01), with a peak at 1-2 h, after CEI and 3-fold (P less than 0.02) in response to CEI plus P or CEI plus PI. P did not block the fall in acid-activated inactive renin compared to CEI alone. The nadir of the inactive renin response to both CEI or CEI plus P occurred at 1-2 h. PI, however, prevented the fall in inactive renin. To extend this observation, we compared the effects of infusion of a vasodilator PG (PGA1; 0.6 micrograms/kg X min) and a pure beta-agonist (isoproterenol; 0.3 micrograms/kg X min). PGA1 increased active renin 2.5-fold and decreased inactive renin by 80% (both P less than 0.02), while isoproterenol increased active renin 4.1-fold, but did not significantly change inactive renin. These data suggest that the beta-adrenergic system and PGs at least acutely stimulate renin production at different steps of its biosynthesis or secretion.

Adult↗
PubMed1983

Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 4 degrees C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we found that in kidney inactive renin constituted from 10 to as much as 50% of the total renin concentration. Biochemical characterization of the inactive renin from plasma and from kidney indicates that they are structural homologues and, when activated, have enzymatic properties that resemble active renal renin. Renal and plasma inactive renin were found to have the following properties in common: (a) a pH optimum of activation of 3.3; (b) reversible activation by acid dialysis on return to pH 7.4, 37 degrees C; (c) pH optima of enzyme activity of 7.8 with sheep angiotensinogen and 5.5 and 6.7 (biphasic) with human angiotensinogen; (d) Michaelis-Menten constants, Km, of 0.29-0.34 microM with sheep angiotensinogen, and 0.99-1.25 microM with human angiotensinogen; (e) an antibody to human renal renin mean inhibitory titer of 1:30,000 with 1 X 10(-4) Goldblatt units of activated renal or plasma inactive renin; (f) gel filtration profiles consisting of two peaks with apparent molecular weights of 56,000 +/- 1,500 and 49,200 +/- 1,000. Activation of plasma and kidney inactive renin by acid plus renal kallikrein was not accompanied by a change in gel filtration elution patterns. To determine whether inactive renin is released by the kidney, we measured inactive renin in samples obtained simultaneously from both the renal veins and inferior vena cava below the origin of the renal veins. In eight consecutive patients, inactive renin concentration was significantly higher in renal venous blood than in inferior vena caval blood. These data indicate that human kidney contains and secretes significant quantities of inactive renin. Thus, the kidney appears to be a major source of inactive renin in human plasma.

Animals↗
PubMed1983

Reciprocal changes in active and inactive renin after converting enzyme inhibition in normal man.

Since one mechanism by which converting enzyme inhibition (CEI) increases renin is removal of angiotensin II negative feedback on the juxtaglomerular cell, we studied the time course of changes in active and inactive renin after CEI. After equilibration on a 25 meq/day sodium diet, captopril was given as a single 50-mg oral dose (acute phase), and then was administered as 50 mg every 6 h for 3 days to seven normal volunteers (chronic phase). In the acute phase, supine blood pressure fell 12 +/- 2 mm Hg (P less than 0.02). Active renin acutely increased 12.5 +/- 0.9 times the baseline value, peaking at 3-4 h. Inactive renin, measured by acid activation of trypsin activation, decreased in all subjects to 10% or less of control from 2 to as long as 6 h post-CEI and then returned to baseline levels by 8 h (P less than 0.01). With chronic CEI, active renin was elevated to 10.8 +/- 2.4 times the baseline level, and after 48 h inactive renin levels rose to 4.0 +/- 0.6 times the baseline (P less than 0.02). To determine whether the acute changes in inactive and active renin occurred because of captopril's effect on renin in the circulation or kidney, a single dose of captopril was administered to three subjects with mild to moderate renal insufficiency and hyporeninemic hypoaldosteronism. In contrast to normal subjects, these patients had no change in active and inactive renin levels when given captopril, suggesting that changes observed in the normals were renal mediated rather than a plasma phenomenon. We conclude that CEI 1) acutely increases active renin while reciprocally reducing the inactive form, and 2) chronically increases both active and inactive renin. These studies support the hypothesis that inactive renin may be a precursor of circulating active renin.

Adult↗
PubMed1982

Changes in active and inactive renin throughout pregnancy.

In the first trimester of pregnancy, inactive renin in plasma rapidly increases (to 5 times the average concentration in plasma of nonpregnant controls), then declines slowly until midpregnancy, and falls quickly to the normal range after delivery. Inactive renin has the same large apparent molecular weight in pregnancy as in control plasma. Amniotic fluid contains very high levels of inactive renin; its mobility on Sephadex G-100 is the same as that of inactive plasma renin, but a lower molecular weight is indicated by the delayed elution of inactive renin of amniotic fluid from Sephacryl S-200. This anomalous behavior is probably responsible for the different estimates of molecular is probably responsible for the different estimates of molecular weight previously reported. The plasma concentration of active renin in pregnancy is modestly increased in the first trimester, declining gradually until term, and falling quickly after delivery. Although the increased PRA in early pregnancy involves an increase in active renin, increased angiotensinogen appears to play a more important part in sustaining the increased PRA of late pregnancy. The apparent molecular weight of te active renin in pregnancy plasma is larger than that in normal plasma. Gross changes in sodium intake during pregnancy result in changes in active and inactive renin concentrations parallel to those observed in nonpregnant controls. These responses suggest that the kidneys are an important source of the altered plasma renin in pregnancy, but do not exclude a contribution from other sources.

Amniotic Fluid↗
PubMed1982

Characterization studies of inactive renin from human kidney and from several plasma sources.

Plasma of normal pregnant women and plasma of some patients with diabetic nephropathy contain increased quantities of an inactive form of renin compared with normal plasma. In order to evaluate the significance of the increased levels of inactive renin in these conditions, we isolated and compared inactive renin from several plasma sources and from human kidney. Renal inactive renin and inactive renin from normal plasma and plasma of patients with diabetic nephropathy and plasma of pregnant women displayed reversible acid activation and similar binding characteristics to cibacron-blue. Using cibacron-blue affinity chromatography, we obtained a totally inactive renin preparation from renal cortex and from plasma of subjects from each of the clinical states. Gel filtration of these preparations and detection of inactive renin using a modified acid activation technique indicated that human inactive renin exists in at least two forms with the following apparent molecular weights: 56,300 +/- 1,500 and 49,200 +/- 1,000 for renal inactive renin, 58,000 and 49,000 for inactive renin in normal plasma, 58,500 and 52,000 for inactive renin in diabetic plasma, 57,000 and 49,000 for inactive renin in pregnancy plasma. These studies suggest that: 1) the kidney is a major source of circulating inactive renin in man, 2) inactive renin from normal, diabetic and pregnancy plasma is similar, and 3) human inactive renin is heterogeneous.

Cold Temperature↗
PubMed1980

Renin requires a structural alteration prior to activation by renal kallikrein.

Renal kallikrein may be the physiologic protease which activates renin. In vitro activation of plasma inactive renin by renal kallikrein, however, requires prior dialysis of plasma to pH 3.3, which is thought to destroy endogenous plasma kallikrein inhibitors. We present evidence that low pH directly alters renin to facilitate its activation by renal kallikrein. Untreated normal plasma was combined with pH 3.3 dialyzed and neutralized plasma and incubated with semipurified human renal kallikrein 2.3 X 10(-2) to 4.6 IU/ml, pH 7.5, 25 degrees C for 1h. All concentrations activated renin in acid-treated plasma; whereas, in untreated plasma 4.6 IU/ml renal kallikrein was ineffective. In combinations of untreated and acid-treated plasma, the percent renin activated by renal kallikrein was equal to the percent acid-dialyzed plasma present. When acid-treated plasma was combined with buffer, activation was complete and quantitatively the same as in the untreated and acid-treated plasma combinations. The data suggest inactive renin itself is altered structurally by pH 3.3; this change in the renin molecule is necessary for kallikrein activation. Renal kallikrein may not be the sole in vivo activator of renin.

Chemical Phenomena↗