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Black or African American↗
Biomedical subjects
M L Johnson
Publications and source records attributed to M L Johnson.
Resolution of end-to-end diffusion coefficients and distance distributions of flexible molecules using fluorescent donor-acceptor and donor-quencher pairs.
We used time-dependent fluorescence energy transfer, time-dependent collisional quenching, and global analysis of the data resulting from these through-space and contact interactions to recover the end-to-end distance distributions and diffusion coefficients of flexible fluorescent molecules. The fluorescence decays of covalently linked tryptamine-acceptor and tryptamine-quencher pairs were measured by the frequency-domain method. These data were fit using numerical solutions of the differential equation, which predicts the time- and distance-dependent population of the excited state donors in the presence of energy transfer or collisional quenching, followed by transformation to the frequency domain for nonlinear least-squares comparison with the experimental data. We found that the energy transfer data for the donor-acceptor pair alone were adequate to recover the starting distribution and the end-to-end diffusion coefficient; however, the resolution is dramatically improved by the use of both the through-space and contact interactions.
Mathematics↗
Distance distributions and anisotropy decays of troponin C and its complex with troponin I.
We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of the two-domain protein, respectively. We developed a procedure to correct for the fraction of the sample that was incompletely labeled with the acceptor independent of chemical data. At pH 7.5 and in the presence of Mg2+, the mean distance was near 15 A with a half-width of the distribution of 15 A; when Mg2+ was replaced by Ca2+, the mean distance increased to 22 A with a decrease in the half-width by 4 A. Similar but less pronounced differences in the mean distance and half-width between samples containing Mg2+ and Ca2+ were also observed with troponin C complexed to troponin I. The results suggest that the conformation of troponin C is altered by Ca2+ binding to the Ca(2+)-specific sites and displacing bound Mg2+ at the Ca2+/Mg2+ sites. This alteration may play an important role in Ca2+ signaling in muscle. At pH 7.5, the anisotropy decays of the donor-labeled troponin C showed two components, with the long rotational correlation time (12 ns) reflecting the overall motion of the protein. When the pH was lowered from 7.5 to 5.2, the mean distribution distance of apotroponin C increased from 22 to 32 A and the half-width decreased by a factor of 2 from 13 to 7 A. The long correlation time of apotroponin C increased to 19 ns at the acidic pH. These results are discussed in terms of a model in which skeletal troponin C is a dimer at low pH and enable comparison of the solution conformation of the protein at neutral pH with a crystal structure obtained at pH 5.2. While the conformation of the monomeric unit of troponin C dimer at pH 5.2 is extended and consistent with the crystal structure, the conformation at neutral pH is likely more compact than the crystal structure predicts.
Animals↗
Analyzing pulsatile endocrine data in patients with chronic renal failure: a brief review of deconvolution techniques.
Deconvolution analysis provides an important new technique to evaluate underlying hormone secretory rates quantitatively based upon serially measured plasma hormone concentrations with or without prior knowledge of the half-time of hormone disappearance from the blood. Information about endocrine gland secretion is particularly important in chronic renal failure, wherein the decreased metabolic clearance rates of various hormones would otherwise confound the interpretation of plasma hormone concentrations. Here we review two particularly useful techniques of deconvolution, one of which is a waveform-defined algorithm and the other waveform independent. The first method can be used to estimate both hormone half-life and secretory rates in vivo. The second methodology allows calculation of in vivo hormone secretion rates without assuming any special form for the secretion event, but requires a priori knowledge of hormone half-life. We illustrate examples of these two deconvolution approaches, and discuss why the interpretations of hormone concentration measurements in earlier studies (where deconvolution methods were not employed) must be viewed with caution. Based on such considerations, additional investigations of in vivo hormone secretory pathophysiology will be required in children and adults with chronic renal failure.
Endocrine Glands↗
Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry.
Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.
Algorithms↗
MY4 expression on B-lymphocyte malignancies may be associated with a more adverse prognosis.
We have noted what other preliminary reports have also described as a new specificity of the MY4 monoclonal antibody from Coulter Immunology which previously was designated to only have myelogenous CD14 specificity. The MY4 marker appears to characterize a subpopulation of some B-lymphocytic malignancies that are CD19, CD20, surface immunoglobulin as well as MY4 positive. The results occurred when other myelogenous markers such as CD11b, CD13 and CD33 were unreactive. Another monoclonal antibody marker of CD14 specificity, MO2, did not demonstrate a similar reactivity. Various other monoclonal antibodies of the same IgGI subclass as MY4 were also not reactive and thereby excludes non-specific adsorption as an explanation of the results. The six patients described in this report represented five non-Hodgkin's lymphoma cases and one chronic lymphocytic leukemia case. Fifteen B-lymphocytic leukemias and 30 other B-lymphocytic non-Hodgkin's lymphomas analysed during the same period were not MY4 positive. In reviewing the clinical course of the six patients compared to the general behavior of these types of malignancies and making a speculative generalization from the small group of cases, the MY4 antigen appeared to be expressed by low to intermediate grade B-lymphocytic malignancies of a type that were more rapidly progressive and/or had a greater tendency to undergo a transformation of their malignancy. Two of the three transformed cases were proceeded by chronic lymphocytic leukemia.
Adult↗
Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry.
We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.
Mathematics↗
Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry.
The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.
Cysteine↗
Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.
The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.
Fourier Analysis↗
Neuroendocrine alterations in the somatotrophic axis in chronic renal failure.
Both secretion and metabolic clearance of GH in health and disease are subject to regulation by an array of metabolic, nutritional, physical activity, age, and body composition cues. Moreover, both GH and its mediators (e.g. IGF-I) are bound in plasma by one or more high-affinity binding proteins. Accordingly, carefully designed clinical and basic investigations are required to examine and clarify the diverse alterations in the dynamics of the somatotrophic axis that accompany the syndrome of CRF with its attendant attenuation of normal anabolism, growth and development.
Carrier Proteins↗
Temporal structure of in vivo growth hormone secretory events in humans.
The time course of spontaneous growth hormone (GH) secretion was studied in 12 normal men by analyzing serum GH concentrations measured in blood collected at 5-min intervals over 24 h with a multiple-parameter deconvolution model to simultaneously resolve endogenous GH secretory and clearance rates. Twenty-four-hour profiles of serum GH concentrations were accounted for by an average of 12 +/- 1.2 (SE) discrete GH secretory bursts having a mean half-duration (duration at half-maximal amplitude) of 25 +/- 2.3 min, indicating that 95% of daily GH secretion occurred in 8.8 h. The majority (96%) of GH was secreted in volleys composed of multiple (4.0 +/- 0.4) discrete secretory bursts. Such volleys of GH secretion were separated by 171 +/- 19 min, whereas their constituent individual secretory events occurred every 36 +/- 1.7 min (P = 0.0001). Between secretory volleys, calculated GH secretory rates fell asymptotically to zero. Significant positive and negative autocorrelations were observed for the series consisting of successive GH secretory burst mass and interburst intervals, respectively. Linear regression analysis revealed significant negative correlations between the mass of GH secreted per burst and both the preceding and following interburst intervals. Estimates of the half-life of endogenous GH (17 +/- 1.7 min) and of the endogenous GH production rate (0.25 +/- 0.033 mg/m2 for an assumed distribution volume of 7.9% body wt) agreed well with earlier independent measurements. We conclude that the human pituitary gland secretes GH in volleys consisting of multiple secretory bursts, without measurable intervening tonic secretion. This pattern of in vivo GH release in normal humans is consistent with a model of high-frequency GH-releasing hormone secretory events superimposed on low-frequency episodes of somatostatin withdrawal.
Adult↗
Biochemical and molecular characterization of variant pyruvate kinase enzymes and genes from three patients with red blood cell pyruvate kinase deficiency.
Pyruvate kinase (PK) from red blood cells (RBC) of three patients with nonspherocytic hemolytic anemia due to PK deficiency was characterized according to internationally standardized methods. The variant enzymes, which were designated PK 'Memphis', PK 'Bartlett', and PK 'Pontotoc', had 11, 60, and 61%, respectively, of the normal enzyme activity. All variant PK enzymes had increased thermolability. Compared with control, Km (PEP) were 200-300% greater for PK 'Memphis', 50% less for PK 'Bartlett' and 300-400% greater for PK 'Pontotoc'. The Km (ADP) were 40 and 300% greater than normal for PK 'Bartlett' and PK 'Pontotoc', respectively. All variants required higher than normal concentrations of the allosteric modifier, fructose-1,6-diphosphate, to achieve 50% activation of maximal enzyme activity. To define the molecular basis of the gene defect, DNA samples from these patients were examined for restriction-fragment-linked polymorphisms. No differences were observed in the structure of the patients' PK genes compared with a normal control. These results are consistent with a mutation in coding sequences, rather than a large insertion, deletion or rearrangement of genetic information, as the underlying genetic defect that accounts for the altered enzyme properties in these PK-deficient patients.
Enzyme Stability↗
Separate sex-influenced and genetic components in spontaneously hypertensive rat hypertension.
Previous results from our laboratory indicated two major genetic components of spontaneously hypertensive rat (SHR) hypertension, an autosomal component and a Y chromosome component. Two new substrains, SHR/a and SHR/y, were developed using a series of backcrosses to isolate each of these components. The SHR/a substrain has the autosomal loci and X chromosome from the SHR strain and the Y chromosome from the Wistar-Kyoto (WKY) rat strain. The SHR/y substrain has only the Y chromosome from the SHR and autosomal loci and X chromosome from the WKY strain. Throughout these breeding programs parents were chosen at random without selection for blood pressure. Males of both substrains maintained blood pressures over 180 mm Hg. Comparisons of blood pressure in these new substrains with the original parental strains can be used to determine the relative proportions of each genetic component in hypertension. The Y chromosome component contributes 34 mm Hg, which is the difference between SHR/y male and WKY male blood pressure. The total autosomal component contributes 46 mm Hg, which is the difference between SHR/a male and WKY male blood pressure. The autosomal component is a sex-influenced trait; males in the SHR/a strain have significantly higher pressures than SHR/a females. Of the 46 mm Hg estimated for the autosomal component, 41 mm Hg is the result of these loci interacting with male phenotypic sex. This sex-influenced component is separate and distinct from the Y chromosome component.
Animals↗
Androgen receptor and the testes influence hypertension in a hybrid rat model.
The objective of this study was to determine if males with a deficient androgen receptor would develop hypertension when crossed with a hypertensive parent. Female King-Holtzman rats (n = 15), heterozygous for the testicular feminization (Tfm) gene, were crossed with male spontaneously hypertensive rats (SHR), and blood pressure was measured weekly from 5-14 weeks in the F1 hybrid males. Approximately 50% of the F1 hybrid males were Tfm males and androgen receptor-deficient, and 50% were normal. Blood pressure in the parent King-Holtzman males, Tfms, and female rats was also followed for the same time period. The F1 normal male hybrids had a significantly higher (p less than 0.05) systolic blood pressure than the Tfm hybrid males after 12 weeks (195 +/- 8 versus 170 +/- 8 mm Hg, respectively). Blood pressure in the male and Tfm Holtzman rats was 120 +/- 5 mm Hg and 110 +/- 6 mm Hg, respectively. Castration lowered blood pressure by 38 mm Hg in the hybrid males and 27 mm Hg in the Tfm hybrids. Female F1 hybrids also showed a pressure rise above that of female Holtzman controls (155 +/- 6 mm Hg versus 110 +/- 6 mm Hg, p less than 0.01) but lower than the F1 males and Tfm hybrids. Ovariectomized females with testosterone implants did not show an elevation in blood pressure. Plasma electrolytes, norepinephrine, and cholesterol were not significantly different between normal and Tfm hybrid males. The results suggest that the presence of an androgen receptor and a testis-derived factor mediate the blood pressure rise in the hybrid males.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vivo biological validation and biophysical modeling of the sensitivity and positive accuracy of endocrine peak detection. II. The follicle-stimulating hormone pulse signal.
Despite interest in the in vivo control of gonadotropin release, valid assessment of the physiological regulation of the pulsatile secretion of the gonadotropin FSH has been hampered by the uncertain validity and reliability of available FSH peak detection algorithms. Difficulties in identifying FSH peaks accurately are believed to arise in part because of the slow metabolic clearance of this glycoprotein hormone. Here, we have used two complementary strategies to test the validity of FSH pulse detection. First, by means of a computer-assisted mathematical model for simulating episodic hormone secretion, we evaluated the effects of various putative FSH secretory pulse amplitudes and half-lives on the sensitivity and positive accuracy of peak detection. Secondly, we used an in vivo primate animal model, in which presumptively true FSH pulses were evaluated independently by continuous electrophysiological monitoring of mediobasal hypothalamic multiunit activity. These two approaches allowed us to define optimal pulse analysis parameters that yield maximal sensitivity and positive accuracy for detecting FSH peaks in synthetic and biological time series. We found (as predicted intuitively) that increasing half-times of hormone disappearance decrease both the sensitivity and positive accuracy of peak detection for any given peak detection thresholds and hormone secretory amplitudes. However, adequately sampled episodic FSH time series could be analyzed for FSH pulsatility by an appropriately constrained, objective computerized algorithm with reasonable (less than 10-15%) false negative and false positive errors, such that resultant sensitivity and positive accuracy exceed 85-90%. Of interest, computer simulations and the in vivo animal model exhibited similar discriminative capabilities. We conclude that increasing half-times of hormone (e.g. FSH) removal do impair hormone peak detection sensitivity and positive accuracy. Nevertheless, gonadotropin time series can be analyzed for FSH pulsatility in a valid manner with adequately constrained false negative and false positive error rates.
Animals↗
Nature of altered growth hormone secretion in hyperthyroidism.
Hyperthyroidism is accompanied by various neuroendocrine regulatory disturbances that affect not only the thyrotropic, but also the gonadotropic, corticotropic, and somatotropic axes. To examine the nature of alterations in neuroendocrine control mechanisms that direct the somatotropic axis in hyperthyroidism, we have applied a novel deconvolution technique designed to estimate the number, amplitude, and mass of significant underlying GH secretory events after the influence of GH metabolic clearance has been removed mathematically. To this end, blood was sampled at 10-min intervals for 24 h in seven hyperthyroid and seven age-matched euthyroid men. The subsequent GH time series were assayed by immunoradiometric assay (sensitivity, 0.08 ng/mL) and submitted to quantitative deconvolution analysis. We found that hyperthyroid compared to euthyroid men 1) had significantly more GH secretory bursts per 24 h (viz. 15 +/- 1.0 vs. 10 +/- 1.1; P = 0.017); 2) secreted 3 times as much GH per burst (3.7 +/- 0.80 vs. 1.3 +/- 0.42 ng/mL distribution vol; P = 0.013); 3) achieved a maximal rate of GH secretion in each burst 2.3-fold higher than that in control men (0.14 +/- 0.028 vs. 0.060 +/- 0.015 ng/mL.min; P = 0.017); and 4) had 3.7-fold higher 24-h endogenous GH production rates (P less than 0.01). Neither hyperthyroid nor euthyroid men had significant interburst (tonic) GH secretion. We conclude that the somatotropic axis in hyperthyroid men is marked by a higher frequency of spontaneous GH secretory bursts, a higher rate of maximal GH secretion attained per burst, and a larger mass of GH released per burst. These neuroregulatory disturbances result in a nearly 4-fold increase in the 24-h production rate of GH in thyrotoxicosis.
Adult↗
Dual defects in pulsatile growth hormone secretion and clearance subserve the hyposomatotropism of obesity in man.
We have examined the mechanisms underlying reduced circulating GH concentrations in the obese human. Computer-assisted (deconvolution) analysis was used to determine endogenous GH secretory and clearance rates quantitatively from entire 24-h plasma GH concentration profiles. These analyses revealed that the half-life (t 1/2) of endogenous GH was significantly shorter in obese (11.7 +/- 1.6 min) than in normal weight subjects (15.5 +/- 0.81 min; P less than 0.01). The accelerated blood disposal rate of GH was not due to decreased circulating concentrations of GH-binding protein, since the latter were similar in obese (25 +/- 1.0%) and normal weight (24 +/- 2.3%) men. However, obese men had significantly fewer GH secretory bursts (3.2 +/- 0.53 vs. 9.7 +/- 0.67/day; P less than 0.01). Among the rare GH secretory bursts that occurred in obese subjects, there were significantly prolonged mean intersecretory burst intervals (282 +/- 65 vs. 131 +/- 11 min; P less than 0.05). The resultant daily GH production rate in obese men was reduced to one fourth that in normal weight individuals. Both GH secretion rate and burst frequency were negatively correlated with the degree of obesity (ponderal index). The decreases in GH burst frequency and half-life were specific, since GH secretory pulse amplitude (maximal rate of GH release), the mass of GH released per burst, and the duration of computer-resolved GH secretory bursts were not different in obese and normal weight men. We conclude that obese men harbor a double defect in GH dynamics involving both GH secretion and clearance, and that the severity of the GH secretory deficiency is proportionate to the degree of obesity.
Adult↗
Analysis of the copulsatility of anterior pituitary hormones.
We have used combinatorial algebra and computer simulations to calculate expected means, variances, and probabilities of hormone-peak coincidences in 2 or more endocrine pulse series. We illustrate application of this conditional probability analysis to pulsatile LH, FSH, and/or PRL data. We observed that 1) serum LH and FSH pulses in 14 young men were randomly associated on different days (P greater than 0.10), but highly synchronized on any given day (P less than 0.0001); 2) serum LH and FSH (P less than 0.0001), LH and PRL (P = 0.023), and FSH and PRL (P = 0.0003) peaks were significantly coupled in healthy postmenopausal women; and 3) the number of triple coincidences among LH, FSH, and PRL release episodes in postmenopausal women significantly exceeded chance expectations (P less than 0.0001). We conclude that suitable statistical coincidence analysis can offer an informative tool with which to evaluate nonrandom event concordance in endocrine investigations, such as clinical studies of the temporally coordinated release of anterior pituitary hormones.
Adult↗