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Biomedical subjects

K E Johnson

Publications and source records attributed to K E Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1991

Improvement of cyclosporin absorption in children after liver transplantation by means of water-soluble vitamin E.

Many childhood recipients of liver transplantation require massive doses of cyclosporin to achieve therapeutic blood concentrations of the drug. The impaired absorption of this strongly lipophilic drug may be due to reduced intestinal absorptive area, suboptimal mixing of the drug with hepatobiliary secretions, or residual cholestasis. Improvement of cyclosporin absorption was sought by means of oral coadministration of d-alpha-tocopheryl-polyethylene-glycol-1000 succinate (TPGS), a water-soluble form of vitamin E which can form micelles. 25 mg/kg daily of TPGS was given to six paediatric liver transplant recipients and one young adult with severe hepatobiliary graft-vs-host disease after bone-marrow transplantation, who required 29-136 mg/kg cyclosporin daily to achieve therapeutic cyclosporin blood concentrations. Five responded; the oral cyclosporin dose could be reduced by 40-72% within 2 months. In addition, intravenous cyclosporin was stopped in two of the responders. In the two non-responders the cyclosporin doses at entry were similar to those in the responders after TPGS treatment. Oral cyclosporin absorption tests correctly predicted the outcome of treatment in three responders and one non-responder tested. Treatment with TPGS to enhance cyclosporin absorption might be a useful way of reducing the high cost of immunosuppression in paediatric liver transplant recipients.

Adult↗
PubMed1991

Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity.

Segments critical to the activity of human granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified by scanning deletion analysis and compared with the critical regions previously identified in the homologous mouse GM-CSF protein. Three of the four critical regions thus identified are in equivalent positions in their respective polypeptides, while a fourth critical region of each is uniquely located. To investigate whether unique critical regions are responsible for the observed species specificity of human and mouse GM-CSF, all critical regions were substituted into their opposite homologue. This identified one specific, but different, critical region in each homologue that could not be replaced. Further characterization of the nature of the species specificity of these two proteins was accomplished by the generation of a series of human/mouse GM-CSF hybrids. Each hybrid protein was assayed for specific activity on human- and mouse GM-CSF-dependent cell lines. Significant differences in the specific activity of these hybrids was observed, suggesting that different segments of each molecule interact with their respective receptors. Based on these two approaches, individual amino acids were identified that could provide, at least in part, the interactions between these protein ligands and their respective receptors. These residues are Thr-78 and Met-80 in human GM-CSF and Asp-92, Thr-98, and Asp-102 in mouse GM-CSF.

Amino Acid Sequence↗
PubMed1991

Mammalian cAMP-dependent protein kinase functionally replaces its homolog in yeast.

The cDNA encoding the catalytic subunit (C alpha) from mouse cAMP-dependent protein kinase (PK) was expressed in Saccharomyces cerevisiae. By a plasmid swap procedure, we demonstrated that the mammalian C alpha subunit can functionally replace its yeast homolog to maintain the viability of a yeast strain containing genetic disruptions of the three TPK genes encoding the yeast C subunits. C alpha subunit produced in yeast was purified and its biochemical properties were determined. The protein isolated from yeast appears to be myristylated, as has been found for C subunits from higher eukaryotic cells. This system would be useful for studying the biochemistry of the mammalian enzyme in vitro and its biological role in a model in vivo system. These studies demonstrate that the PK substrate(s) required for viability are recognized by the mammalian enzyme. In general terms, these results demonstrate that heterologous proteins with only 50% sequence conservation with their yeast counterparts can be functional in yeast. This is an important result because it validates the use of yeast to identify the biological role of newly cloned genes from heterologous systems, a key tenet of the Human Genome Initiative.

Amino Acid Sequence↗
PubMed1991

Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI.

We have demonstrated the existence of human cytokine synthesis inhibitory factor (CSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BCRFI. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this assay, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

Amino Acid Sequence↗
PubMed1991

Mechanical circulatory assistance after heart transplantation.

From October 1985 through December 1989, 92 heart transplant procedures were performed in 89 patients. Nine patients (aged 19 to 66 years; 7 male, 2 female) required mechanical circulatory support after transplantation because of primary idiopathic organ failure (n = 2), implant difficulty (2), poor organ quality (2), or acute right heart failure (3). Devices used included the intraaortic balloon pump (6), centrifugal right ventricular assist device (2), left ventricular assist (1), biventricular assists (2), and total artificial heart (1). Two patients required multiple devices. One patient underwent retransplantation. Implant time ranged from 1 to 18 days. One early death occurred owing to right heart failure 6 days after transplantation, 7 hours after removal of a right ventricular assist device, for an overall mortality of 11%. The remaining 8 patients are alive 4 months to 28 months after transplantation. The actuarial 1-year survival of 89% +/- 10% compares well with the survival of 87% +/- 4% for the entire transplant group. All surviving patients are in functional class I. Echocardiographic examination in all patients revealed left ventricular ejection fraction to be normal in 7 and depressed in 1. Extending the criteria for organ donors or difficulty with the implant procedure can lead to early organ failure, which may be reversible with circulatory assistance allowing excellent survival.

Adult↗
PubMed1991

Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in Saccharomyces cerevisiae.

The heterologous expression of protein kinases in E. coli has proved difficult and unpredictable. Although the v-abl protein kinase is successfully expressed in E. coli, our experiments on expression of yeast C subunits in E. coli produced large amounts of predominantly insoluble and inactive protein. Attempts to refold the protein proved unsuccessful. In contrast, a major fraction of mouse C alpha expressed in E. coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble. In addition, the E. coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems. Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E. coli. However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function. In addition, baculovirus expression is not particularly useful for studying biological questions. Mouse C alpha and C beta have been overexpressed in NIH3T3 cells. This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background. This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type. Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies. Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished. This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast. We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart. Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit. Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol Dehydrogenase↗
PubMed1991

Electrophysiologic evaluation of cardiovascular agents in the isolated intact rabbit heart.

A modification of the Langendorff perfused rabbit heart is described. This model can be used to perform electrophysiologic studies in the isolated heart and is ideally suited for acute pharmacologic evaluation. His bundle electrograms were measured with a plunge electrode and allowed atrioventricular (AV) nodal physiology to be directly and precisely evaluated. Atrial conduction and refractoriness, atrioventricular node conduction and refractoriness, His-Purkinje conduction, and ventricular conduction and refractoriness could be accurately measured. Verapamil had a depressant effect on AV nodal conduction and refractoriness without affecting His--Purkinje conduction or myocardial refractoriness. Flecainide prolonged atrial conduction and His-Purkinje conduction with less effect on AV nodal physiology. The use of this isolated perfused rabbit heart model is an inexpensive and reliable technique for the evaluation of the electrophysiologic effects of pharmacologic agents on the conduction system of the heart. A major advantage of the isolated heart model is the absence of autonomic reflexes, which can confound studies performed in intact animals.

Animals↗
PubMed1991

Electrophysiologic effects of verapamil metabolites in the isolated heart.

The electrophysiologic effects of the metabolites of verapamil are unknown and may contribute to the observed differences between intravenous and oral verapamil. We examined the electrophysiologic effects of verapamil and its metabolites (norverapamil, N-dealkylverapamil (D617), and N-dealkylnorverapamil (D620)) at estimated, free therapeutic concentrations, in the retrogradely perfused, isolated rabbit heart. Verapamil at 5 and 10 ng/ml significantly prolonged anterograde (11 and 27%, respectively) and retrograde (10 and 25%, respectively) atrioventricular (AV) nodal block cycle lengths. Anterograde and retrograde AV nodal conduction times and refractory periods were also prolonged. Norverapamil at 100 ng/ml had qualitatively similar effects equivalent to 20-50% that observed with verapamil at 10 ng/ml. D620 had small but statistically significant effects on some AV nodal parameters. D617 had no effect. The combination of verapamil plus its principal metabolite, norverapamil, had additive effects. None of the compounds had any measurable effect on atrial conduction, His-Purkinje conduction, or atrial refractoriness. Ventricular refractoriness was significantly prolonged only by norverapamil. In conclusion, some of the metabolites of verapamil have important electrophysiologic AV nodal effects and may contribute to the clinical effects observed during chronic oral verapamil dosing.

Animals↗
PubMed1991

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.

Acinetobacter calcoaceticus↗
PubMed1990

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male.

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.

Animals↗
PubMed1990

In vivo analyses of integrin beta 1 subunit function in fibronectin matrix assembly.

Early development of the urodele amphibian Pleurodeles waltl is accompanied by a process of progressive fibronectin (FN) fibrillogenesis. FN begins to assemble into fibrils on the inner surface of the blastocoele roof at the early blastula stage and progressively forms a complex extracellular matrix. We have analyzed the mechanisms of FN-fibril formation under normal and experimental conditions in vivo with the following probes: iodinated FN, fluorescein-labeled FN, synthetic peptides containing the Arg-Gly-Asp (RGD) cell surface recognition sequence of FN, and polyclonal antibodies against both beta 1 subunit of the amphibian FN receptor and the cytoplasmic domain of beta 1 subunit. We report that in living embryos, exogenous labeled mammalian FN injected into the amphibian blastocoele undergoes FN-fibril formation in spatiotemporal patterns similar to those of endogenous FN. This indicates regulation of fibrillogenesis by the cell surface rather than by changes in the type of FN. Fibrillogenesis is inhibited in a dose-dependent manner both by the GRGDS peptide and monospecific antibodies to amphibian integrin beta 1 subunit. Furthermore, when injected intracellularly into uncleaved embryos or into selected blastomeres, antibodies to the cytoplasmic domain of integrin beta 1 subunit produce a reversible inhibition of FN-fibril formation that follows early cell lineages and cause delays in development. Together, these data indicate that in vivo, the integrin beta 1 subunit and the RGD recognition signal are essential for the proper assembly of FN fibrils in early amphibian development.

Amino Acid Sequence↗
PubMed1990

Fibronectin-rich fibrillar extracellular matrix controls cell migration during amphibian gastrulation.

We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.

Amphibians↗
PubMed1989

Cells from Rana pipiens gastrulae and arrested hybrid gastrulae show differences in adhesion to fibronectin-sepharose beads.

Experiments were performed to examine adhesion of Rana pipiens gastrula cells and arrested hybrid gastrula cells to fibronectin-Sepharose beads (FN-beads). Blastula cells from both normal and hybrid embryos show poor adhesion to FN-beads. Beginning at the early gastrula stage, however, normal cells show a progressively increasing tendency to adhere to beads. In two different arrested hybrid embryos, cells from all developmental stages lack the ability to adhere to beads. A third hybrid shows an increase and then a decrease in cell-bead adhesion. A fourth hybrid shows a late increase in cell-bead adhesion in animal-half cells and no increase at all in vegetal-half cells. Blastula-stage cells have the ability to adhere to con A-beads and two kinds of Cytodex beads but will not adhere to FN-beads. Similarly, some cells from arrested hybrid embryos lack the ability to adhere to FN-beads but will adhere to con A-beads and cytodex beads. Observations in the light and scanning electron microscope show that normal cells form lamellipodia on FN-beads and move about actively on them, much like they do in vivo on surfaces coated by fibrils containing fibronectin. For adherent hybrid cells attached to beads, one kind does so by small pseudopodia but does not move on them and another kind forms active lamellipodia at the tips of fusiform cells and moves on beads.

Animals↗
PubMed1989

Community-based health care in Kibwezi, Kenya: 10 years in retrospect.

In response to the interest of the Kenya government in community-based health care, the Kibwezi Rural Health Scheme was developed by the African Medical and Research Foundation (AMREF) in a semi-arid district in eastern Kenya. Based on a community co-operative philosophy and focussing on health promotion and prevention, the scheme includes the following: a health centre with a 15-bed in-patient unit including four maternity beds, out-patient services, and a 15-bed nutrition rehabilitation unit; a cadre of volunteer community health workers, trained by AMREF, who form the backbone of the project; maternal child health/family planning and nutrition services including an applied nutrition programme, a water project; and a mobile health unit. Designed as a replicable model health programme, the intention was that services would be gradually taken over by the Ministry of Health of Kenya. Much has been learned in the development of the project which should be meaningful to others considering similar endeavours. One of the first lessons learned was that the time taken to sensitize the community to community-based health care is critical to the success of the project and may need to be as long as 1-2 years. Another was that gaining the support of the community for the community health workers (CHW) requires a considerable effort on the part of project staff, but seems to be the only viable solution to the remuneration and recognition of the CHW's work. It also became apparent that preventive and promotive health services should be integrated structurally and operationally with curative health services to provide the most benefits for the community served. Finally, although there are some differences of opinion, it is felt that with some refinements, the project could be replicated in other parts of Kenya.

Child Health Services↗
PubMed1989

Results of the first 100 patients who received Symbion Total Artificial Hearts as a bridge to cardiac transplantation.

Between August 8, 1985 and April 1, 1988, 100 patients in 22 centers underwent Symbion Total Artificial Heart (TAH) (Symbion, Inc., Salt Lake City, Utah) implantation as a bridge to cardiac transplant. Thirty patients received the Symbion J7-100, and 70 patients received Symbion J7-70 implants. Eighty-seven patients were men and 13 were women; mean age was 42 years, and mean weight was 73 kg. The time spent on the device ranged from 1 to 243 days, for a mean of 23 days. Sixty-eight patients underwent cardiac transplantation, with a 69% 30-day survival rate and a 46% (31 of 68) long-term survival rate. The majority of the patients had ischemic (52) or idiopathic (36) cardiomyopathies. Indications for implantation of the TAH occurred most frequently in patients whose condition deteriorated while awaiting transplant (34) or who were admitted in acute cardiogenic shock (27). There was an 8.7% incidence of thromboembolic events (four cerebrovascular accidents, five transient ischemic attacks). Multivariant analysis was performed to determine the effect of various factors on the patients' ability to undergo transplantation and then ultimate survival. The results indicate a need for further clinical investigation in patient selection and appropriate timing for implantation of the TAH.

Adult↗