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Biomedical subjects

R G Johnson

Publications and source records attributed to R G Johnson.

At least 19 recordsLinked to original sources
PubMed2003

The association between milk urea nitrogen and DHI production variables in western commercial dairy herds.

A retrospective observational study was conducted using data from Dairy Herd Improvement monthly tests to investigate the association between milk urea nitrogen (MUN) concentration and milk yield, milk protein, milk fat percentage, SCC, and parity for commercial Holstein and Jersey herds in Utah, Idaho, and Montana. Mean MUN for Holstein cows was 15.5 mg/ dl (5.5 mmol/L) MUN and 14.1 mg/dl (5.0 mmol/L) for Jersey cows. Mean MUN, categorized by 30-d increments of days in milk (DIM), paralleled changes in milk values and followed a curvilinear shape. For Holstein cows, concentrations of MUN were different among lactation groups 1, 2, and 3+ for the first 90 DIM for Holsteins. Overall, concentrations of MUN were lower during for the first 30 DIM compared with all other DIM categories for both Holstein and Jersey cows. Multivariate regression models of MUN by milk protein showed that as the milk protein percentage increased, MUN concentration decreased; however, models for Jersey cows showed that MUN did not decrease significantly until above 3.4% milk protein. Milk fat percentage also decreased as MUN increased, but by only 1 mg/dl MUN over the range of 2.2 to 5.8% milk fat. Somatic cell count showed a negative relationship with MUN. Holstein cows with milk protein percentage >3.2% had lower MUN compared with cows having milk protein <3.2% for milk yields from 27.3 to 54.5 kg/d and lower than cows having a milk protein <3.0% for milk yield of 54.5 to 63.6 kg/d. In Jersey cows, MUN concentrations were not different among milk protein percentage categorized by milk yield. This study found that MUN was inversely associated with milk protein percentage and paralleled change in milk yield over time.

Animals↗
PubMed2001

Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Animals↗
PubMed2001

Effects of L-749,329, an ET(A)/ET(B) endothelin receptor antagonist, in a porcine coronary artery injury model of vascular restenosis.

BACKGROUND: Previous studies in animal models of angioplasty have suggested a role in neointimal hyperplasia for endothelins (ETs), potent vasoconstricting peptides that also exert growth-promoting effects. The present studies were undertaken to test the hypothesis that endothelin receptor blockade can reduce neointimal thickening in injured porcine coronary arteries. METHODS AND RESULTS: An ET(A)/ET(B) antagonist, L-749,329, was evaluated as an inhibitor of intimal thickening in a porcine balloon/stent model of coronary artery injury. L-749,329 competitively inhibited [(125)I]ET-1 binding to porcine ET(A) (IC(50) approximately 0.3 nmol/L) or ET(B) (IC(50) approximately 20 nmol/L) receptors and inhibited ET-1-stimulated signaling in cell culture. In anesthetized pigs, big ET-1-stimulated increases in systemic blood pressure were totally inhibited after intravenous infusion of L-749,329 (>/=0.2 mg. kg(-1). h(-1)). In vascular injury studies, pigs were treated with vehicle or L-749,329 (1 mg. kg(-1). h(-1)) beginning 2 days before and continuing 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of an angioplasty balloon wrapped with a coiled metallic stent. After 28 days, mean neointimal thickness in the L-749,329-treated group was reduced by 9.0% compared with vehicle-treated controls, but this effect was not statistically significant (P=0.13). CONCLUSIONS: Blockade of endothelin receptors for 28 days with only a mixed ET(A)/ET(B) receptor antagonist is insufficient to substantially inhibit intimal hyperplasia after balloon/stent coronary artery injury in the pig, in contrast to results with a selective ET(A) antagonist. The effects of selective or mixed ET(A)/ET(B) antagonists in diseased vessels remain to be determined in this model.

Acetamides↗
PubMed2001

Risk analysis in patients bridged to transplantation.

BACKGROUND: Efforts to predict mortality in bridge to cardiac transplant patients have concentrated on preventricular assist device (VAD) status. To more fully identify factors influencing survival to transplant, we reviewed the preoperative and postoperative VAD courses of 105 bridge to transplant patients. METHODS: Sixty-four parameters (34 pre-VAD, 30 post-VAD), including hemodynamics, complications, and evaluations of major organ function were examined and analyzed. RESULTS: Thirty-three patients (31%) died on VADs and 72 were transplanted. There were two posttransplant operative deaths (3%). By univariate analysis 23 of 64 factors were significant. These 23 factors were entered into a stepwise logistic regression analysis to identify predictors of survival to transplant. Four factors, including pre-VAD intubation (p < 0.005), cardiopulmonary bypass (CPB) time during VAD insertion (p < 0.0001), mean pulmonary artery pressure (first postoperative day after VAD) (p < 0.0002), and highest post-VAD creatinine (p < 0.01) were independent predictors of transplantation. CONCLUSIONS: Other than the need for intubation, pre-VAD variables were of little value in predicting survival to transplant. Problems during VAD insertion (long CPB time) and post-VAD renal insufficiency were independent predictors. Severe complications that developed during the interval of VAD support, including cerebrovascular accident, bleeding and infection, were surprisingly not predictors for transplantation.

Actuarial Analysis↗
PubMed2001

Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells.

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two G(i alpha) proteins (G(i alpha 2) and G(i alpha 3)), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

Adenosine Diphosphate Ribose↗
PubMed2000

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Animals↗
PubMed2000

Characterization and quantitation of phospholamban and its phosphorylation state using antibodies.

Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.

Animals↗
PubMed2000

The effects of aprotinin on blood product transfusion associated with thoracic aortic surgery requiring deep hypothermic circulatory arrest.

OBJECTIVE: To compare the effects of aprotinin on blood product use and postoperative complications in patients undergoing thoracic aortic surgery requiring deep hypothermic circulatory arrest. DESIGN: A retrospective study. SETTING: A university hospital. PARTICIPANTS: Nineteen patients who underwent elective or urgent thoracic aortic surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The total number of units of packed red blood cells, fresh frozen plasma, and platelets was significantly less in the group that received aprotinin (p = 0.01, 0.04, and 0.01). The intraoperative transfusion of packed red blood cells and platelets, collection and retransfusion of cell saver, and postoperative transfusion of fresh frozen plasma were also significantly less in the aprotinin group (p = 0.01, 0.02, 0.01, and 0.05). No patient in either group sustained renal dysfunction or a myocardial infarction. Two patients who had not received aprotinin suffered from chronic postoperative seizures, and one patient who had received aprotinin sustained a perioperative stroke. CONCLUSIONS: Low-dose aprotinin administration significantly decreases blood product transfusion requirements in the setting of thoracic aortic surgery requiring deep hypothermic circulatory arrest, and it does not appear to be associated with renal or myocardial dysfunction.

Aorta, Thoracic↗
PubMed2000

Postoperative atrial tachyarrhythmias in patients undergoing coronary artery bypass graft surgery without cardiopulmonary bypass: a role for intraoperative magnesium supplementation.

OBJECTIVE: To determine if intraoperative magnesium supplementation would be associated with a reduction in postoperative atrial tachyarrhythmias (POAT) in patients undergoing coronary artery bypass grafting (CABG) surgery without cardiopulmonary bypass (off-pump CABG surgery). DESIGN: Retrospective study. SETTING: University Medical Center. PARTICIPANTS: Patients who had undergone off-pump CABG surgery (n = 124). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The charts of 124 patients who had undergone off-pump CABG surgery (64 by anterior thoracotomy and 60 by median sternotomy) were retrospectively reviewed. Demographic data and perioperative care were recorded and compared among patients who did and did not experience POAT and among patients who did and did not receive intraoperative magnesium supplementation. Logistic regression analysis was used to assess the association between magnesium supplementation and incidence of POAT, controlling for other covariables. Of the 124 patients, 16 had a prior history of atrial or ventricular arrhythmias and/or were receiving antiarrhythmic medications. Medical records of the remaining 108 patients were reviewed. Twenty-four patients (22%) had POAT. Forty-two patients (39%) received intraoperative magnesium. In patients receiving intraoperative magnesium, the incidence of POAT was significantly decreased (12% v 29%; p = 0.03). In these patients, initial postoperative serum magnesium was significantly higher (2.37 mEq/L v 1.86 mEq/L; p < 0.01). In patients not receiving intraoperative magnesium, 35% had hypomagnesemia (serum magnesium < 1.8 mEq/L) compared with 9% of patients receiving magnesium (p < 0.01). Patients who received intraoperative magnesium and beta-adrenergic blockers had a lower incidence of POAT (5%) than patients who received only one (19%) or neither (33%) (p < 0.05). CONCLUSIONS: Intraoperative magnesium supplementation is associated with a decrease in POAT after off-pump CABG surgery. The combination of a beta-blocker and magnesium may reduce POAT further. It is recommended that intraoperative magnesium supplementation be part of the care of patients undergoing off-pump CABG surgery.

Adrenergic beta-Antagonists↗
PubMed2000

The impact of foodborne calicivirus disease: the Minnesota experience.

The first outbreaks of Norwalk virus gastroenteritis in Minnesota were confirmed in 1982. Since then, Norwalk-like caliciviruses have been recognized to be the most common cause of foodborne disease outbreaks, accounting for 41% of all confirmed foodborne outbreaks in Minnesota from 1981-1998. Although laboratory confirmation of caliciviruses in stool samples was not attempted in most of these outbreaks, all conformed to epidemiologic criteria for defining outbreaks of Norwalk virus. Since 1996, the availability of polymerase chain reaction testing at the Minnesota Department of Health has allowed for the confirmation of calicivirus infection among patients involved in epidemiologically defined outbreaks of viral gastroenteritis. Results have confirmed the usefulness of characterizing foodborne disease outbreaks by epidemiologic criteria and also confirmed the importance of human caliciviruses as the leading cause of foodborne disease outbreaks in Minnesota.

Caliciviridae Infections↗
PubMed2000

Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

1-Methyl-3-isobutylxanthine↗
PubMed2000

A novel approach for assessing the quality and effectiveness of IACUC oversight in investigator compliance.

In research facilities that are registered with the U.S. Department of Agriculture (USDA), funded by the Public Health Service, or accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, the Institutional Animal Care and Use Committee (IACUC) is charged with oversight and evaluation of animal care and use under the terms of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Although the committee's oversight of investigator compliance may be evaluated annually during USDA inspections and triennially during AAALAC International site visits, routinely assessing the quality and effectiveness of the IACUC's performance is difficult. To measure the successfulness of IACUC oversight, our committee retained a management consultant to objectively design and conduct a confidential survey that could be used to determine how the IACUC could improve the process of facilitating researcher compliance with federal regulations and accreditation standards. The consultant based the content of the survey on confidential interviews with all IACUC members, the IACUC administrator, and a cross sectional representation of the key animal-user population at the facility. The survey was then distributed to the entire animal-user population. Vice-presidents, directors, principal investigators, and technicians were included in the distribution. With a response rate of 34%, the survey results indicated that the facilitation process warranted refinements. The consultant provided the IACUC with its recommendations, which were based on the discernible trending information indicated in the survey responses. The IACUC developed a specific plan of action to address the consultant's recommendations and intends to re-survey the animal-user population once the action plan has been fully implemented. In summary, the survey is an excellent way to assess the quality and effectiveness of IACUC oversight in investigator compliance by determining the level of researcher satisfaction. The evaluation, review, and follow-up process using a confidential interview and questionnaire technique can enhance the performance and effectiveness of IACUC oversight.

Accreditation↗
PubMed1999

Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase.

A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.

Adenosine Triphosphate↗
PubMed1999

Effects of isoproterenol on twitch contraction of wild type and phospholamban-deficient murine ventricular myocardium.

Ablation of the gene for phospholamban (PLB), a transmembrane peptide regulator for the cardiac sarcoplasmic reticulum Ca2+ pump, in mice brings about a complete loss of the myocardial responses to beta-adrenergic agonists (e.g., Luo et al., Circ. Res. 1994; 75: 401). We have evaluated the functional significance of PLB-independent mechanisms in the myocardial responses to beta-adrenergic stimulation in isolated intact ventricular myocardium. We compared the effects of (-)-isoproterenol (ISO) on isometric twitch contraction of paced right ventricular muscle strips of wild type (WT) and PLB-deficient (PLBKO) mice. At 37 degrees C, frequent spontaneous contractions in both types of muscles required the inclusion of lidocaine, an antiarrhythmic, in the bathing medium. Thus the experiments were also performed at two lower temperatures, 30 degrees C and 25 degrees C, at which lidocaine was not needed. Under three conditions, in the absence of ISO, PLBKO ventricular muscles exhibited substantially shortened time to peak tension (TPT) and half relaxation time (TR1/2), compared with the WT muscles. In both WT and PLBKO muscles ISO increased the peak developed tension and decreased TPT and TR1/2 in a dose-dependent manner although the effects were generally smaller in PLBKO than in WT muscles. One micromolar ISO caused TPT and TR1/2 to decrease by 7.3+/-1.2% (mean+/-SEM) and 7.5+/-1.2% in PLBKO vs. 22.8+/-0.7% and 29.1+/-1.7% in WT at 37 degrees C; by 13.5+/-0.4% and 14.1+/-1.2% in PLBKO vs. 31.3+/-0.8%, and 44.8+/-1.3% in WT at 30 degrees C; by 15.0+/-2.3% and 21.1+/-4.9% in PLBKO vs. 25.8+/-1.9% and 54.0+/-1.9% in WT at 25 degrees C. These findings strongly suggest that PLB-independent mechanisms play a significant role in mediating the positive inotropic and lusitropic effects of beta-adrenergic agonists on ventricular myocardium.

Adrenergic Agonists↗