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J D Johnson

Publications and source records attributed to J D Johnson.

At least 289 records · Page 16Linked to original sources

The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+.

We have modeled the time-course of Ca2+ binding to calmodulin, troponin, parvalbumin, and myosin in response to trains of transient increases in the free myoplasmic calcium ion concentration (pCa). A simple mathematical expression was used to describe each pCa transient, the shape and duration of which is qualitatively similar to those thought to occur in vivo. These calculations assumed that all individual metal binding sites are noninteracting and that Ca2+ bind competitively to the Ca2+-Mg2+ sites of troponin, parvalbumin, and myosin. All the on-and-off rate constants for both Ca2+ and Mg2+ were obtained either from the literature or from our own research. The percent saturation of the Ca2+-Mg2+ sites with Ca2+ was found to change very little in response to each pCa transient in the presence of 2.5 X 10(-3)M Mg2+. Our analysis suggests that the Ca2+ content of these sites is a measure of the intensity and frequency of recent muscle activity because large changes in the Ca2+ occupancy of these sites can occur with repeated stimulation. In contrast, large rapid changes in the amount of Ca2+ bound to the Ca2+-specific sites of troponin and calmodulin are induced by each pCa transient. Thus, only sites of the "Ca2+-specific" type can act as rapid Ca2+-regulatory sites in muscle. Fluctuation in the total amount of Ca2+ bound to these sites in response to various types of pCa transients further suggests that in vivo only about one-half to one-third of the total steady-state myofibrillar Ca2+-binding capacity exchanges Ca2+ during any single transient.

Animals↗

Two types of ventilated porometers compared on broadleaf and coniferous species.

Two ventilated porometers (diffusion and steady-state) were compared on four broadleaf and five coniferous species. The diffusion porometer gave consistently lower conductance values for both types of species, reflecting a direct stomatal response to low chamber humidity. At high conductance values, the porometers produced a linear and nearly equal response, but the diffusion porometer was less sensitive at low conductance values. This was due to lower air flow (20% of the velocity in the steady-state porometer) and water vapor sorption (by its acrylic plastic chamber). The broadleaf species had less variation (R(2) = 0.81) than did the coniferous species (R(2) = 0.61), but, with the latter, there was better correspondence between the two porometers, possibly due to sampling technique. Conductance values were clustered by species.

Journal Article↗

Catabolism of human very low density lipoproteins in vitro: a fluorescent phospholipid method for monitoring lipolysis.

The catabolism of human plasma very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase has been measured in vitro using a fluorescent phospholipid as a method to monitor lipolysis. Dansyl phosphatidylethanolamine (DPE) was incorporated into VLDL to form DPE-VLDL, and the rate of catabolism was followed by measuring the increase in fluorescence at 490 nm after the addition of the enzyme. The studies were performed with VLDL isolated from 20 normal individuals. In addition, the VLDL from 8 mildly obese subjects with primary hypertriglyceridemia (Type IV phenotype) was studied. With this in vitro system and with a constant amount of lipoprotein lipase, the rate of lipolysis did not differ in normal and in these hypertriglyceridemic subjects. Furthermore, there was no correlation between the rates of hydrolysis and the plasma levels of triglyceride or high density lipoprotein cholesterol.

Adult↗

Hydrolysis of guinea pig nascent very low density lipoproteins catalyzed by lipoprotein lipase: activation by hjman apolipoprotein C-II.

Very low density lipoproteins isolated from guinea pig liver perfusate (VLDLp) lack the equivalent of human apolipoprotein C-II (apoC-II), the activator of lipoprotein lipase (LpL). These lipoproteins are therefore ideal substrates with which to investigate the mechanism by which apoC-II activates the enzyme. VLDLp binds apoC-II, and apoC-II associated with VLDLp markedly increases the rate of lipoprotein lipase-catalyzed hydrolysis of VLDLp-triglycerides. The activator potency of apoC-II is independent of the method of enrichment of VLDLp with apoC-II: delipidated human apoC-II and apoC-II transferred from human high density lipoproteins activate lipoprotein lipase to equal extents. ApoC-II causes pH-dependent changes in both apparent Km and VmaX of LpL-catalyzed hydrolysis of VLDLp-triglycerides. At pH l7.4--7.5, the major effects of apoC-II is to decrease the apparent Km by 3.3--4.0 fold. The apparent Vmax is increased 1.3-fold. At pH 6.5 and 8.5, the decrease of apparent Km is less marked, 1.6-fold and 1.4-fold, respectively. At pH 6.5, apoC-II increases the apparent Vmax ty 1.3-fold, while at pH 8.5 the primary effect of apoC-II is a 1.6-fold increase of apparent Vmax. Based on a simple kinetic model, the data suggest that apoC-II favors direct interaction between enzyme and triglyceride within the lipoprotein particle, as well as subsequent catalytic turnover.

Animals↗

Magnesium and the regulation of muscle contraction.

There are a variety of Ca2+ binding sites in muscle (e.g., troponin, parvalbumin, myosin, and calmodulin) that may play a role in the regulation of muscle contraction an other enzymatic processes. since most of these proteins also bind Mg2+, it is important to consider the effect that the high free Mg2+ concentration (mM) found in muscle has on the Ca2+ binding properties of these sites. The major effect of Mg2+ is to greatly reduce the rate of Ca2+ binding to the sites that bind Mg2+ and Ca2+ competitively (Ca2+-Mg2+-type sites found in troponin, parvalbumin, and myosin, which would be essentially saturated with Mg2+ in a relaxed muscle) due to the slow dissociation of bound Mg2+. Thus during a transient increase in [Ca2+] similar to that which would occur during muscle activation, these sites would bind very little Ca2+ and, consequently, could not play a regulatory role. Even if Ca2+ were able to bind to these sites during muscle activation (e.g., if the free Mg2+ in muscle is lower than presumed), the dissociation of Ca2+ from these sites would be quite slow due to their very slow off rates for Ca2+, again making these sites unsuitable for participating in a rapid Ca2+-induced switching mechanisms. In contrast, the Ca2+-specific-type (regulatory) sites found in troponin and calmodulin do not have these restraints. The rate of Ca2+ binding to these sites is not affected by Mg2+ and the off rate of Ca2+ from these sites is very rapid due to their lower Ca2+ affinity. Thus, these sites are able to respond to rapid Ca2+ transients, an essential feature for any Ca2+ binding site that plays a regulator role.

Binding Sites↗

A double blind study of the effects of oral indomethacin in preterm infants with patent ductus arteriosus who failed medical management.

Over a two year period, 52 infants were found to have clinical signs of patent ductus arteriosus (PDA). Twenty-seven responded to fluid restriction and furosemide; the remaining 25 infants entered the Indomethacin (IN) study protocol. Their mean (+/- SE) gestational age was 29.3 (+/- 0.6) weeks and birth weight was 1,142 (+/- 80) gm. Either a placebo or IN (0.25 mg/kg) orally was given for two doses, 24 hours apart; if no response occurred, the patient was crossed over to the opposite medication. Using Chi-square analysis, a significant response rate to IN was found. There were no significant differences in birth weights, gestational ages, or fluid intake between responders and nonresponders. However, both responders and nonresponders required a prolonged ventilator course, suggesting factors other than PDA causing prolonged ventilatory requirements in these babies.

Double-Blind Method↗

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.

Adenosine Triphosphatases↗

A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C.

Cardiac troponin C (C-TnC) was labeled with the sulfhydryl-specific fluorescent probe molecule 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid at cysteine 35 and 84 to produce C-TnCIA. This modified protein binds Ca2+, undergoes Ca2+-induced increases in alpha helix, and forms a complex with other troponin subunits as does unlabeled C-TnC. C-TnCIA undergoes a small fluorescence decrease with Ca2+ or Mg2+ binding to the two high affinity Ca2+-Mg2+ sites of C-TnC and a large biphasic approximately 2.1-fold fluorescence increase with Ca2+ binding to two lower affinity Ca2+-specific sites with KCa of approximately 4.5 X 10(5) M-1 and approximately 5 X 10(2) M-1. C-TnCIA was formed in a complex with troponin I (TnI) and troponin T to form C-TnIA. This fluorescent reconstituted whole troponin undergoes a 25% decrease with Ca2+ binding to a Ca2+-specific site of KCa approximately 3 X 10(6) M-1. C-TnC, therefore, contains a single Ca2+-specific site of approximate equal affinity as the two Ca2+-specific regulatory sites of skeletal TnC. This Ca2+-specific site in C-TnC (like its two corresponding sites in S-TnC) undergoes an approximate 10-fold increase in affinity in whole troponin or when TnC is complexed with TnI. Since the two Ca2+-specific sites in skeletal troponin have been shown to be the regulatory sites of skeletal muscle contraction we suggest that this single Ca2+-specific site, of equal affinity, in C-TnC is the regulatory site of cardiac muscle contraction.

Animals↗

Incorporation of dipalmitoyl phosphatidycholine into human plasma low density lipoproteins: effects on composition and structure.

Bovine liver phosphatidylcholine exchange protein was used to transfer di[14C]palmitoyl phosphatidylcholine from sonicated vesicles to human plasma low density lipoproteins (LDL). The incorporation of di[14C]-palmitoyl phosphatidylcholine into LDL was associated with an increased percentage of total phospholipid and increased lipid rigidity as shown by an increase in the fluorescence polarization of 12-(9-anthroyloxy)-stearic acid and 1,6-diphenyl-1,3,5-hexatriene.

Animals↗

Effects of 5-fluorouracil on the formation of modified nucleosides in yeast transfer RNA.

Yeast cells grown in the presence of the drug FUra synthesize RNA in which Urd is partially replaced by FUrd. Transfer RNAs in which 1.5-50% of the Urd has been replaced by FUrd have been isolated and their base compositions measured to determine the effect of FUrd incorporation on posttranscriptional nucleoside modification. This replacement results in an extensive reduction in the amounts of Thd, H56Urd and psird found in mature tRNA. Quantitatively, the reduction of psird greater than or equal to Thd greater than H56Urd. The losses of psird, Thd and H56Urd are greater than can be accounted for by the stoichiometry of FUrd incorporation. The formation of 5-MeCyd is not affected by the drug, whereas the methylated purines show substoichiometric losses in FUrd-containing tRNAs. In Escherichia coli, we have not observed any effects of FUra on the methylated purine content, although the effects on psird, Thd and H56Urd are similar. These findings indicate that (a) in both pro- and eukaryotic systems the enzymes responsible for psird, Thd and H56Urd formation are affected by FUra treatment in a similar manner; (b) prokaryotic purine methylases may be more tolerant of structural aberrations resulting from FUrd incorporation than eukaryotic methylases and (c) different methylases within one system show different sensitivities as shown by those responsible for 1-MeAdo and 5-MeCyd formation.

Base Composition↗

Dansyl phosphatidylethanolamine-labeled very low density lipoproteins. A fluorescent probe for monitoring lipolysis.

The fluorescent phospholipid dansyl phosphatidylethanolamine (DPE) (dansyl, 5-dimethylaminonaphthalene-1-sulfonyl) was incorporated into very low density lipoproteins (VLDL) to form DPE-VLDL. The addition of milk lipoprotein lipase to DPE-VLDL in the presence of albumin resulted in a greater than 3-fold fluorescence increase and a 20 nm blue shift in the wavelength of the emission maxima of the dansyl fluorophore. The lipoprotein lipase-induced fluorescence changes occurred concomitantly with the release of free fatty acids from VLDL. Lipoprotein lipase did not produce fluorescence changes in DPE incorporated into either low or high density lipoproteins. The rate of fluorescence increase in DPE-VLDL was maximal at 37 degrees C, dependent on the concentration of lipoprotein lipase and VLDL, and followed typical Michaelis-Menten kinetics with a Km of 1.0 for lipoprotein lipase. Both the initial rate and the total fluorescence increase correlated well (r = 0.98 and 0.95) with the release of free fatty acids. We conclude that the lipoprotein lipase-induced fluorescence increases in DPE-VLDL provide an accurate, convenient, and the only noninvasive means of following continuously the lipolysis of human VLDL.

Animals↗

Clinical role of the mixed cell agglutination test in the prospective management of superficial bladder tumors.

The mixed cell agglutination test allows the selection of patients with transitional cell carcinoma of the bladder who are at risk to develop invasive disease. A review of our retrospective experience and that of others reveals an overall accuracy of 78% in the prediction of tumor behavior. Current prospective studies are described and a rational, selective approach to the follow-up of bladder tumor patients, influenced by the MCA test result, is advocated.

Antigens, Neoplasm↗

Intranasal flunisolide in the treatment of perennial rhinitis: Correlation with immunologic parameters.

The effectiveness and safety of 200 micrograms/day of intranasal flunisolide in the treatment of perennial rhinitis was studied in 56 patients in a 6 wk double-blind parallel vehicle controlled clinical trial. In addition, patients failing to respond to placebo were entered into a 6 wk open trial with the active drug. Forty-six percent of the flunisolide-treated patients achieved total or substantial control of their nasal symptoms compared to 11% of the placebo-treated group in the double-blind study (p = 0.031). Eighty percent of patients achieved total or substantial control of their nasal symptoms in the 6 wk open study. No adverse effects attributable to flunisolide were observed. Parameters of IgE-mediated reactivity, including immediate-type skin test reactivity, total serum and nasal secretion IgE, specific serum and nasal secretion IgE, and nasal eosinophilia, were also assessed in these patients. Although benefit from flunisolide significantly correlated with all of these parameters except specific serum IgE, the absence of these findings did not preclude significant benefit from the drug. This study demonstrates the efficacy and safety of intranasal flunisolide in the treatment of perennial rhinitis, especially but not exclusively in those patients with evidence of IgE-mediated reactivity.

Administration, Intranasal↗