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Biomedical subjects

K E Johnson

Publications and source records attributed to K E Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1989

Mechanical circulatory support as a bridge to transplantation.

From December 1985 through April 1988, 11 patients (three female and eight males), 33.1 +/- 3.9 (+/- SEM) years of age (range, 15 to 50 years), underwent heart transplantation preceded by the use of mechanical circulatory support. The causes of cardiac failure were ischemic (four), viral cardiomyopathy (three), idiopathic cardiomyopathy (two), congenital heart disease (one), and valvular heart disease (one). All patients were preterminal. Mechanical circulatory support consisted of intra-aortic balloon pump (eight), the total artificial heart (seven), biventricular assist (three), and left ventricular assist (two). Seven patients had more than one form of support. The duration of mechanical circulatory support was 12.2 +/- 4.1 days (range, 1 to 44 days). Once listed for transplantation, patients waited for 8.1 +/- 2.4 days for a donor. Seven patients received OKT3 monoclonal antibody as prophylaxis, in addition to triple-drug immunosuppression. There were four rejection episodes and 12 serious infections. In addition, eight patients suffered a major posttransplant complication of a distant organ system: central nervous system (three), renal (two), and respiratory (three). Post transplant hospitalization was 48 +/- 22 days (range, 15 to 248 days). Two patients (18%) died after transplant, one of severe acute rejection (29 days) and one of multisystem failure (248 days). All patients surviving transplant hospitalization are alive 6 to 34 months after the transplant procedure. Eight patients are in functional class I and 1 patient class II. This patient attends school full-time but has a premechanical support system neurologic defect. In follow-up of 163 patient-months except for yearly catheterization, these nine patients required only 5 hospital days. Although the use of the mechanical circulatory support as a bridge to transplantation can result in a prolonged, complicated hospitalization after transplant, the long-term results are gratifying.

Adolescent↗
PubMed1988

Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1.

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.

Carrier Proteins↗
PubMed1988

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit.

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.

Amino Acid Sequence↗
PubMed1988

A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation.

A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.

Amino Acid Sequence↗
PubMed1988

The 140-kDa fibronectin receptor complex is required for mesodermal cell adhesion during gastrulation in the amphibian Pleurodeles waltlii.

We have studied the localization and function of a 140-kDa glycoprotein complex implicated in cell adhesion to fibronectin- and laminin-rich extracellular matrices in Pleurodeles waltlii gastrulae. In particular, we have shown that antibodies directed against highly purified avian fibronectin (FN) receptor complex cross-react with two major polypeptides of apparent molecular weights of 140,000 and 100,000 and a third minor component of 90,000. Using sections of embryos or whole mounts, we have also discovered that the putative FN receptor is widely distributed on the early embryonic cell surface. We have also found that the basal surface of the roof of the blastocoel, a region particularly enriched in an extracellular matrix consisting of fibronectin- and laminin-rich fibrils, is rich in receptor complex. We have prepared monovalent Fab' fragments of this antibody and have found that they cause detachment of cells previously attached to substrata coated with fibronectin, and they also arrest gastrulation when injected into the blastocoel of early gastrulae. Thus, it appears that the fibronectin receptor complex plays a significant functional role in cell attachment of gastrula-stage cells in vitro and in cell migration in vivo during gastrulation.

Animals↗
PubMed1987

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization.

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.

Base Sequence↗
PubMed1986

Nitrobenzyl derivatives as bioreductive alkylating agents: evidence for the reductive formation of a reactive intermediate.

o- and p-nitrobenzyl chlorides and carbamates were chemically and electrochemically reduced in the presence and absence of the nucleophile morpholine; activation of these compounds by reduction was required to produce an intermediate capable of alkylation. The reduction products formed by the catalytic hydrogenation of each compound were examined by gas chromatography-mass spectrometry. In addition, the products generated by controlled-potential electrolysis were examined by ESR and NMR spectrometry. After a one-electron reduction, o- and p-nitrobenzyl chlorides were activated to the nitrobenzyl radicals, which subsequently dimerized to the dinitrobibenzyl derivatives or reacted with morpholine when present in the reaction medium to form the (nitrobenzyl)morpholine adducts. The nitrobenzyl carbamates were not activated after a one-electron reduction; however, the morpholine and the ether adducts of these agents were observed after catalytic hydrogenation. It was assumed that an intermediate or intermediates formed after the one-electron reduction product, or the full reduction product of the carbamates, were capable of alkylating various nucleophiles. Chemical reduction of the potential bioreductive alkylating agent (o-nitrobenzyl)-6-thioguanine produced (o-aminobenzyl)-6-thioguanine, indicating a lack of formation of a reactive electrophile by reduction. (o-, (m-, and (p-nitrobenzyl)-6-thioguanine analogues were also examined for cytotoxic activity toward EMT6 tumor cells under aerobic and hypoxic conditions. In agreement with the inability of (o-nitrobenzyl)-6-thioguanine to form a reactive species after chemical reduction, no decrease in the survival of neoplastic cells exposed to 10(-4) M drug occurred under either aerobic or hypoxic conditions.

Alkylating Agents↗
PubMed1986

Transplantation studies to investigate mesoderm-ectoderm adhesive cell interactions during gastrulation.

Experiments involving transplantation of the roof of the blastocoel in Rana pipiens embryos reveal that the inner surface of the roof of the blastocoel must be coated with a fibrous extracellular matrix (F-ECM) to serve as a substratum for mesodermal cell migration. When the roof of the blastocoel is inverted the original outer surface, now projecting toward the blastocoel, does not become coated with F-ECM and does not support mesodermal cell migration. When the roof of the blastocoel is removed from a normal embryo and transplanted into an interspecific arrested hybrid embryo known to be deficient in F-ECM synthesis, the grafted ectodermal fragment does not become coated with F-ECM and does not support normal mesodermal cell migration. When a hybrid graft is placed in a normal embryo, the grafted ectodermal fragment becomes coated with F-ECM and supports mesodermal cell migration. In normal control embryos migrating mesodermal cells are polarized due to formation of lamellipodia on their leading but not their trailing edges. These cells are arranged in overlapping layers. The leading cells form lamellipodia on the roof of the blastocoel and trailing cells form lamellipodia on one another.

Animals↗
PubMed1986

Control of day care shigellosis: a trial of convalescent day care in isolation.

Simultaneous outbreaks of S. sonnei infections occurred in September 1983 at two day care centers in Seattle, Washington. At both centers, there were high rates of diarrheal illness in the children (22/80 and 11/23, respectively), in staff members, and in family members of the ill children. The smaller center was temporarily closed, but the larger one remained open. Convalescent children and staff whose diarrhea had resolved and who were receiving appropriate antimicrobials were allowed to return to the larger center before negative cultures were obtained and were separated in an isolation room. No culture-proven cases of shigellosis occurred at either center in the following two months. Plasmid profile determinations suggested the Shigella were not transmitted from the center that remained open to the community. Estimated need for alternate child care requirements at the closed center were 100-fold greater than at the center with the isolation room. Caring for convalescent children in isolation at day care centers while they receive antimicrobial therapy may be a useful strategy for controlling day care shigellosis.

Adult↗
PubMed1986

Diarrhea associated with adherent enteropathogenic Escherichia coli in an infant and toddler center, Seattle, Washington.

During November 1983, the Seattle-King County Department of Public Health investigated an outbreak of diarrhea associated with enteropathogenic Escherichia coli, serogroup 0111:K58, in an infant and toddler day-care center. Of the 25 children in the center, ranging in age from 4 to 30 months (median age 11 months), diarrhea occurred in 14 characterized by watery, greenish stools. The median duration of diarrhea was 12 days. Two of the ill children were hospitalized because of severe dehydration. Stool cultures from the children diagnosed initially did not yield the common bacterial pathogens, parasites, or rotavirus. Stool cultures from 11 of 14 ill children and two of 11 well children (P less than .005), however, yielded an E coli serogroup, 0111:K58, which was not invasive or toxigenic by standard tests. The source of the organism was not identified. Although this organism has been recognized as a cause of diarrhea in newborn nurseries, this is the first published report of a documented outbreak of enteropathogenic E coli-induced diarrhea in a day-care center in the United States.

Child Day Care Centers↗
PubMed1986

Cells from Xenopus laevis gastrulae adhere to fibronectin-sepharose beads and other lectin coated beads.

Cells from Xenopus laevis blastulae have a poorly developed ability to adhere to Sepharose beads covalently coupled to bovine plasma fibronectin (FN-beads). They do, however, have the ability to adhere to con A-beads and cytodex-1 and cytodex-3 beads. Beginning at the early gastrula stage, there is a progressively increasing ability of cells to adhere to FN-beads. Gastrula cells adhere to FN-beads by the formation of large ruffling lamellipodia. These cells can translocate on the surface of FN-beads; and when attached to both beads and the surrounding glass substratum of culture vessels, have the ability to move the beads extensively. Gastrula cells also have the ability to adhere to but not move upon con A-beads, wheat germ agglutinin-beads, and soy bean agglutinin-beads. They do not adhere significantly to Tetragonolobus purpureas agglutinin-beads. These results suggest that there are increasing numbers of fibronectin receptors present on the surface of embryonic amphibian cells during the period of gastrulation. They may explain the differential distribution of fibronectin-containing fibrils in vivo as observed by scanning electron microscopy.

Animals↗
PubMed1985

Community-wide surveillance of Campylobacter jejuni infection. Evaluation of a laboratory-based method.

Surveillance of infections due to Campylobacter jejuni is needed to further define the epidemiology of this disease in the U.S. We assessed one potential method of community-wide surveillance, a laboratory-based reporting network. Six microbiology laboratories that routinely culture fecal specimens for C. jejuni reported all isolates to Seattle-King County Health Department for an 18-month period. Further investigations on 476 cases reported from this broadly based network revealed that C. jejuni enteritis affected primarily children and young adults, occurred sporadically (with a summer-fall predominance), led to hospitalization in 13% of cases, was found negligibly in patients hospitalized or immunocompromised, and produced no fatalities. Comparative statistics showed that C. jejuni was reported more frequently than Salmonella and Shigella combined in individuals served by reporting laboratories. Because one participating laboratory was that of a 280,000-member health maintenance organization, it was possible to calculate age-specific incidence rates for bacterial enteric infections in that population. C. jejuni and Salmonella had high attack rates among 0-4 year-old children: 184 cases/100,000 enrollees per year and 126/100,000 per year, respectively. C. jejuni showed another peak at 20-29 years (108/100,000 per year) that was absent in salmonellosis. This study demonstrates that a laboratory-based reporting system is a feasible approach to community-wide C. jejuni surveillance.

Adolescent↗
PubMed1984

Effects of ethanol on the primitive streak stage mouse embryo.

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.

Animals↗
PubMed1984

Intracellular acidosis blocks the basolateral Na-K pump in rabbit urinary bladder.

The purpose of the experiments described in this paper was to examine the effect of intracellular pH on the basolateral Na+-K+-ATPase of rabbit urinary bladder cells. To modify the intracellular pH, we permeabilized the apical membrane of the bladder cells with the polyene antibiotic nystatin. To verify that the intracellular pH could be altered after nystatin treatment, the intracellular pH was monitored with pH-sensitive microelectrodes. (The normal intracellular pH was 7.1 +/- 0.11, n = 21.) After nystatin treatment, the intracellular pH over the range of pH 5.8 to 8.0 was indistinguishable from the mucosal pH. The cell-to-serosa, ouabain-inhibitable sodium flux showed a strong dependence on intracellular pH, with pH values more acidic or more alkaline than 7.4 producing substantial block. The magnitude of the alkaline block obeyed a sigmoidal relationship with a half block of Na+ flux near pH 8.1. The magnitude of the acidic block also obeyed a sigmoidal relationship with half block at pH 6.85. The combination of the two relationships produced a bell-shaped curve with a maximum between pH 7.3 and 7.6. The steepness of both relationships suggested that H+ ion was interacting with more than one site. At the normal intracellular pH (7.1), the Na pump was already partially blocked. This partial block, coupled with the steepness of the relationship between Na pump activity and intracellular H+ near pH 7.1, suggests that intracellular pH could be an important controlling factor for Na pump activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1984

Ectodermal fragments from normal frog gastrulae condition substrata to support normal and hybrid mesodermal cell migration in vitro.

Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers during gastrulation. They can also be deposited in vitro where they condition substrata in such a way that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos can condition culture substrata so as to promote moderate cell attachment and, for one particular gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested hybrid embryos. These results provide new evidence to support the hypothesis that extracellular fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.

Animals↗
PubMed1984

Circus movements in dissociated cells from two new hybrid frog embryos.

Circus movements are a peculiar kind of cell movement, seen in many amphibian cells, that involve the formation of a hyaline protrusion from the cell surface and rotation of this blister around the circumference of the cell. Studies of cells from normal Rana pipiens embryos have shown a close correlation between circus movements in vitro and gastrulation related cell movements in vivo. Cells from two developmentally arrested hybrid embryos (R. pipiens female X R. sylvatica male and R. pipiens female X R. catesbiana male) are deficient in circus movements to an extent that corresponds to their gastrulation deficiencies. This study reports on the preparation of two new hybrids (R. pipiens female X R. temporaria male (temp) and R. pipiens female X R. clamitans male (clam)). Cells from temp embryos show some circus movements but the proportion of cells displaying such movements does not increase with increasing developmental age to the same extent as is seen in cells from normal embryos. Cells from clam embryos show very few circus movements at any developmental age. These aberrations in the onset and extent of circus movements are discussed in relation to characteristic morphogenetic events that occur in normal embryos and in these two new arrested hybrid embryos.

Animals↗
PubMed1983

Type II collagen-induced arthritis. A morphologic and biochemical study of articular cartilage.

Articular cartilage was obtained from type II collagen-induced arthritic rat joints. Transmission electron microscopy showed a gradual degeneration of chondrocytes, disorganization of the collagenous extracellular matrix, and formation of microscars. Biochemical analyses indicated that type II collagen was the only collagen present and that it was normal in regard to hydroxylation of lysine and glycosylation of hydroxylysine. Analyses of the proteoglycan in the extracellular matrix revealed a 50% loss of chondroitin sulfate and keratan sulfate.

Animals↗