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We describe procedures for determining cytosine and orotic acid in urine. We determine cytosine by cation-exchange analysis with either HCl or pH 5.2 buffer as eluent. Orotic acid is first separated by an anion-exchange separative procedure; after lyophilization, the product is subjected to "high-pressure" liquid chromatography for further separation and detection. We analyzed urine from normal subjects and from immunodeficient children. Three children with severe combined immunodeficiency had increased levels of cytosine in urine (23-160 mmol/mol creatinine); one child with severe combined immunodeficiency and two children with other immunodeficiencies had normal urinary levels (less than 2 mmol/mol creatinine). Orotic acid excretion in urine was normal (1-5 mmol/mol creatinine) in all of th immunodeficient children. We discuss the possible significance of the increased cytosine excretion in the three children with severe combined immunodeficiency. | US |
The great concern over the status and care of the dying cancer patient requires the understanding of current trends in care. An 18-year review of 55,288 death certificates of patients with cancer in Cuyahoga County, Ohio (1957--1974) revealed that 35,381 patients (65%) died in acute and chronic care hospitals, 8,251 patients (15%) died in nursing homes, and 11,242 patients (20%) died at home. Trends over the 18-year period demonstrated a shift from patients dying at home to patients dying in nursing homes. The hospital care of dying cancer patients remained unchanged during the study period. An analysis of 33 consecutive patients dying of cancer over a six-month period in an acute care hospital in Cuyahoga County showed an average length of stay of 20.1 +/- 15.7 days, during which only palliative care was provided. The cost benefit of home care/hospice programs is related to the final hospital stay of the dying cancer patient. | US |
The Hypertension Detection and Follow-up Program (HDFP), a national collaborative study, screened approximately 159,000 people for high blood pressure in 14 communities between 1973 and 1974. Results show that detection, treatment, and control of high blood pressure has improved considerably since the 1960s. Whereas in the past about half of all hypertensives knew they had high blood pressure, half of those detected were under treatment, and half of those under treatment had their high blood pressure under control, the corresponding percentages in the 14 HDFP communities a decade later indicate that 75% of hypertensives were detected, 72% of those were under treatment, and 70% of those under treatment had a diastolic blood pressure under 95 mm Hg. While the differences in prevalence of hypertension are between races rather than sexes (with black individuals in some age groups being about twice as likely as white individuals to have hypertension), the differences in detection and treatment rate are largely between sexes and not between races. Women are considerable more likely to be aware of their hypertension, to be under treatment for it, and to have their high blood pressure under control. Rates of control vary considerably among age-sex-race subgroups, from only 8% of white male hypertensives aged 30-39 to 67% of white female hypertensives aged 60--69. It appears that although efforts to combat this disease over the past decade have probably made considerable progress in improving the recognition and treatment of high blood pressure, there remain a large number of undetected, untreated, and uncontrolled hypertensives. | US |
The end of the collagen fibril is an important and unique morphological site. Growth of fibrils in vitro and in vivo appears to occur by addition of ordered subassemblies of collagen aggregates at the fibril end. In tissues the fibril end appears closely related to the cell surface in both mesenchymal cells and epithelia. In the latter cell type there is an apparent relationship between the basement membrane and fibril end. Platelets do not aggregate upon exposure to basement membranes or collagens derived therefrom. We suggest that the end of the fibril which is embedded in the outer face of the basement membrane may be involved in interaction with the platelet. | US |
1. Human platelets will react with a number of different collagens from guinea-pig to ostrich. 2. Human skin collagen when treated with pepsin does not react with human platelets. 3. Calf platelets display a different pattern of reactivity than that of human platelets. | US |
Bovine rhodopsin and isorhodopsin were excited with a single 530-nm, 7-ps light pulse emitted by a mode-locked Nd 3+ glass laser at room temperature. Within 3 ps of excitation, absorbance changes due to formation of bathorhodopsin were observed. The difference spectra generated during and 100 ps after pulse excitation are presented. The data show that bathorhodopsin formation is completed within 3 ps for both the primary pigments and suggest that a single common bathorhodopsin is photochemically formed from both primary pigments. Our findings provide additional support for the cis-trans isomerization model of the primary event in vision. Additional absorption transients that were observed near 670 and 460 nm are discussed. | US |
To test our present quantitative knowledge of nicotinic transmission, we reconstruct the postsynaptic conductance change that results after a presynaptic nerve terminal liberates a quantum of acetylcholine (ACh) into the synaptic cleft. The theory assumes that ACh appears suddenly in the cleft and that is subsequent fate is determined by radial diffusion, by enzymatic hydrolysis, and by binding to receptors. Each receptor has one channel and two ACh binding sites; the channel opens when both sites are occupied and the rate-limiting step id the binding and dissociation of the second ACh molecule. The calculations reproduce the experimentally measured growth phase (200 microseconds), peak number of open channels (2,000), and exponential decay phase. The time constant of the decay phase exceeds the channel duration by approximately equal to 20%. The normal event is highly localized: at the peak, two-thirds of the open channels are within an area of 0.15 micrometer 2. This represents 75% of the available channels within this area. The model also simulates voltage and temperature dependence and effects of inactivating esterase and receptors. The calculations show that in the absence of esterase, transmitter is buffered by binding to receptors and the postsynaptic response can be potentiated. | US |
This paper investigates the accuracy of the resistive-force theory (Gray and Hancock method) which is commonly used for hydrodynamic analysis of swimming flagella. We made a comparison between the forces, bending moments, and shear moments calculated by resistive-force theory and by the more accurate slender-body theory for large-amplitude, planar wave forms computed for a flagellar model. By making an upward empirical adjustment, by about 35%, of the classical drag coefficient values used in the resistive-force theory calculations, we obtained good agreement between the distributions of the forces and moments along the length of the flagellum predicted by the two methods when the flagellum has no cell body attached. After this adjustment, we found the rate of energy expenditure calculated by the two methods for the few typical test cases to be almost identical. The resistive-force theory is thus completely satisfactory for use in analysis of mechanisms for the control of flagellar bending, at the current level of sophistication of this analysis. We also examined the effects of the presence of a cell body attached to one end of the flagellum, which modifies the flow field experienced by the flagellum. This interaction, which is not considered in resistive-force theory, is probably insignificant for small cell bodies, such as the heads of simple spermatozoa, but for larger cell bodies, or cell bodies that have large-amplitude motions transverse to the swimming direction, use of slender-body theory is required for accurate analysis. | US |
Syncytial tissues consist of many cells whose intracellular spaces are electrically coupled one to another. Such tissues typically include narrow, tortuous extracellular space and often have specialized membranes at their outer surface. We derive differential equations to describe the potentials induced when a sinusoidal or steady current is applied to the intracellular space with a microelectrode. We derive solutions for spherical preparations with isotropic properties or with a particular anisotropy in effective extracellular and intracellular resistivities. Solutions are presented in an approximate form with a simple physical interpretation. The leading term in the intracellular potential describes an "isopotential" cell in which there is no spatial variation of intracellular potential. The leading term in the extracellular potential, and thus the potential across the inner membranes, varies with radial position, even at zero frequency. The next term of the potentials describes the direct effects of the point source of current and, for the parameters given here, acts as a series resistance producing a large local potential drop essentially independent of frequency. A lumped equivalent circuit describes the "low frequency" behavior of the syncytium, and a distributed circuit gives a reasonably accurate general description. Graphs of the spatial variation and frequency dependence of intracellular, extracellular, and transmembrane potential are given, the response to sinusoidal currents is used to calculate numerically the response to a step function of current. | US |
The electrical properties of the crystalline lens of the frog eye are measured with stochastic currents applied with a microelectrode near the center of the preparation and potential recorded just under the surface. The stochastic signals are decomposed by Fourier analysis into sinusoidal components, and the impedance is determined from the ratio of mean cross power to input power. The data are fit by an electrical model that includes two paths for current flow: one through the cytoplasm, gap junctions, and outer membrane; the other through inner membranes and the extracellular space between lens fibers. The electrical properties of the structures of the lens which appear as circuit components in the model are determined by the fit to the data. The resistivity of the extracellular space within the lens is comparable to the resistivity of Ringer. The outer membrane has a normal resistance of 5 kohm . cm(2) but large capacitance of 10 muF/cm(2), probably because it represents the properties of several layers of fibers. The inner membranes have properties reminiscent of artificial lipid bilayers: they have high membrane resistance, 2.2 megohm . cm(2), and low specific capacitance, 0.8 muF/cm(2). There is so much membrane within the lens, however, that the sum of the current flow across all the inner membranes is comparable to that across the outer surface. | US |
A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper. | US |
The intracellular reference phase (RP) method and ultra-low temperature micro-dissection were used for isothermal and isotopic phase distribution studies of Na(+), K(+), and water in amphibian oocyte cytoplasm. One-third of the cytoplasmic water is available as solvent for [(3)H]sucrose. This fraction, designated c1, quantitatively coincides with the water volume in which Na(+) and K(+) are freely diffusible. Two-thirds of the cytoplasmic water is inaccessible to sucrose and is designated c2. The Na(+) and K(+) associated with c2 are extremely slowly exchanging (bound) and at different concentrations than in c1. The cations in c1 are in mass-action equilibria with those in c2, each described by an equation of the formC(c) (i) = C(c) (1) (i) + C(c) (2) (i) = q(i).C(RP) (i) + (max)C(c) (2) (i).f(C(RP) (i)in which C(c) (i) is the cytoplasmic Na(+) or K(+) concentration, C(c) (1) (i) is the free, and C(c) (2) (i) the bound cation concentration averaged over the cytoplasmic water. q(i) is the fractional free solute space, C(RP) (i) the RP concentration, (max)C(c) (2) (i) the concentration of binding sites, and the function f is satisfied by the Langmuir isotherm. Numerical values for the variables of the isotherm are determined. Activity coefficients are calculated from RP data and provide a basis for generalizing the oocyte results to other cells. The conclusion is drawn that both c1 and c2 are widely distributed in cells, and that cellular ionic activities involve two distinct systems: the cell-membrane system and an adsorbed water ion-exchange-like buffering system. Alternative explanations for the two-component cytoplasm are considered. A model is proposed in which c1 is a normal intracellular aqueous phase controlled by the plasma membrane, whereas c2 consists of water and ions adsorbed in hydrate crystalline structures. In oocytes these structures are identified with yolk platelets. | US |
A model of a 100 micrometers diameter Purkinje fiber with intercellular clefts was studied under voltage clamp conditions to examine the consequences of radial nonuniformity. Sodium and potassium conductances were distributed so that the surface and cleft membranes had similar channel density. Assuming that the model is appropriate, sodium current (and conductance) measured in the voltage clamp is grossly underestimated because of loss of voltage control of the cleft membrane. Under these conditions a value for g Na of about 15-20 mmho/cm2 of actual membrane is consistent with the experimental measurements of Dudel and Rüdel (1970. Pfluegers Arch. Eur. J. Physiol. 315:136-158.). Intermediate and slow currents (slow inward current and potassium current) appear to be accurately measured under the model conditions, despite some voltage nonuniformity within the cleft. This result depended on the presence of a residual sodium current, and experimental removal of sodium may alter this result. All effects of nonuniformity would be accentuated in fibers of larger diameter. | US |
It is shown that the constant field approximation must be amended to make it apply to time-dependent signals. The necessary additional term corresponds to the ionic displacement current. In the absence of adsorption, this ionic displacement current is found to have a characteristic time of the order of a fraction of a microsecond. We confirm its mathematical form as given by Cole (1965). When the membrane-soluble ions are strongly adsorbed, an additional, purely exponential transient of much larger time constant is calculated, with a time dependence identical to that of the translocation of adsorbed ions. Our results support the pseudostationary approximation used by Andersen and Fuchs (1975) in the description of such exponential transients. Explicit expressions are given for the current after a voltage step as for the admittance, both in the absence and presence of adsorption, for a membrane with a rectangular potential energy profile. | US |
The effect of uncharged, dipolar phloretin on anion and cation conductance through a black lipid membrane can be used to study its adsorption behavior. The adsorption of phloretin can be described by a Langmuir isotherm with weak dipole-dipole interaction. | US |
The inward sodium current in cardiac muscle is difficult to study by voltage clamp methods, so various indirect experimental measures have been used to obtain insight into its characteristics. These methods depend on the relationship between maximal upstroke velocity of the action potential (Vmax) and the sodium current (INa), usually defined in terms of the Hodgkin-Huxley model. These relationships were explored using an adaptation of this model to cardiac Purkinje fibers. In general Vmax corresponded to INa, and it could be used to determine the relationship of membrane potential to GNa, and h infinity. The results, however, depended on the method of stimulation of the action potential, and an optimal stimulation method was determined. A commonly used experimental technique called "membrane responsiveness" was shown to distort seriously the properties of steady-state gating inactivation that is supposed to measure. Estimation of the changes in maximal sodium conductance, such as those produced by tetrodotoxin (TTX), would be accurately measured. Some experimental results have indicated a voltage-dependent effect of TTX. Characteristics of the measures of TTX effect under those conditions were illustrated. In summary, calculations with a model of the cardiac Purkinje fiber action potential provide insight into the accuracy of certain experimental methods using maximal upstroke velocity as a measure of INa, and cast doubt on other experimental methods, such as membrane responsiveness. | US |
The Raman spectrum of all-trans anhydrovitamin A in hexane at 77 degrees K is presented. The similarity of the Raman spectra of anhydrovitamin A and the protonated Schiff base of retinal is striking. The implications of this for visual pigment studies and bacteriorhodopsin are discussed. Tentative assignments of geometry for four cis-trans isomers of anhydrovitamin A are made on the basis of the observed room-temperature absorption spectra. | US |
Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers. | US |
Experiments to determine the apparent dissociation constants of the Ca and Mg complexes of arsenazo III clearly indicated that the predominant Ca complex contains one Ca ion and two dye molecules, although previous reports have either claimed or assumed 1:1 complexing. The evidence is based on the effects of varying [dye] as well as [Ca] and [Mg], and clear evidence for the formation of 1:1 complexes with Ca was obtained only at submicromolar [dye], whereas Mg formed 1:1 complexes exclusively. The implications of these findings with regard to the use of arsenazo III as an indicator of intracellular free [Ca] are discussed, with particular reference to its selectivity for Ca and the interference effects of other ions. | US |
The effect of pancuronium on alamethicin-induced currents was studied in negatively charged lipid bilayer membranes. Pancuronium induces inactivation of the alamethicin-induced current. Inactivation is only observed if this compound is added to the compartment containing alamethicin. Moreover, the process of inactivation is reduced or abolished if pancuronium is added to the alamethicin-free side of the membrane. The time needed to recover from inactivation is greatly reduced if the aqueous solution in the alamethicin-free compartment is stirred. These data suggest that pancuronium permeates through the membrane when the alamethicin-induced conductance is "turned on," binds to the other membrane surface, and changes the surface potential. | US |
The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values. | US |
A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm. | US |
The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol. | US |
Using a method they developed, Stamatoff and Krimm (1976) have phased swelling data from nerve myelin. Although most phases agree with those I determined previously, there are a few differences. In this letter the two different phasings, theirs and my own, are used to compute the corresponding electron-density profiles, which are then closely compared. For both phasings, small differences are seen in the membrane profile at different degrees of swelling. The explanation that these differences are due simply to errors in measuring intensity is shown to be quite improbable; thus the differences indicate a real change in the profile. It follows that the assumption of a constant membrane profile appears to be invalid in the case of myelin swelling. The differences therefore are assumed to indicate a real change in the profile. It is shown that this change can be attributed consistently to interdigitation of protein molecules at the surfaces of neighboring membranes, while the membrane structure itself remains unchanged. In this case, valid phases still can be determined by swelling, but the phases determined by Stamatoff and Krimm are not valid. | US |
A mathematical model of the fluorescence decay experiment based on linear systems theory is presented. The model suggests an experimental technique that increases the probability of correctly determining the decay constants of a multicomponent system. The use of moment methods for data analysis improves accuracy by combining information obtained from several discrete experiments. Examples are presented to show that the analysis of a three component system composed of known standards is improved as the number of experimental determinations is increased from one to four. The discrete measurements are made by changing the excitation and emission wavelengths. | US |
Light-scattering intensities in the I parallel and I+ mode were obtained on thin sections of three human lenses. Random density and orientation fluctuation theory, without cross correlation, was employed to evaluate light-scattering parameters. Both the density correlation distances, as well as the orientation correlation distances, were related to structural elements in the lens fiber cell that have been observed by other investigators with different techniques. The magnitude of these fluctuations were evaluated, and it was demonstrated that the density fluctuations are the main contributors to light scattering in normal human lenses. Changes in the light-scattering parameters were evaluated as a function of position within the lens. The changes observed agree with the biochemical data in the literature that reflects that an aging process occurs when one proceeds from the periphery of the lens toward the center. | US |
The quenching of fluorescence due to energy transfer between a dilute, random array of donor and acceptor chromophores in lipid bilayer was measured and compared to theoretical expressions developed to predict the decrease in emission intensity under these circumstances. The observed intensity was found to be the same function of quencher concentration in both planar, multilamellar dispersions and small, spherical vesicles. The degree of quenching was accurately predicted by a simple relation derived in this paper, as well as a more complex equation previously developed by Tweet, et al. The results suggest that significant quenching may be observed even when the average donor-acceptor separation exceeds the Förster critical distance by severalfold. Application of these results to problems of current interest in membrane research are discussed. | US |
Two kinetic models are introduced which predict amplitudes and time-courses of endplate currents and miniature endplate currents at neuromuscular junctions, at both normal and acetylcholinesterase-inhibited endplates. Appropriate differential rate equations reflecting interactions of acetylcholine with acetylcholine receptor and with esterase, diffusion of acetylcholine both within and from the synaptic cleft, and cooperativity between receptor site occupancy and ion channel opening are solved. Acetylcholine release into the cleft is assumed to be instantaneous. The simpler homogeneous reaction space model accurately predicts decay phase time constants are inaccurate. The two-reaction space model predicts amplitudes and time constants within a factor of two of those observed experimentally. The simulations indicate that the amplitudes and time-courses are primarily determined by the chemical reaction rates that characterize acetylcholine interactions with receptor and esterase and that these interactions occur under nonequilibrium conditions. Approximately 50% of the total ion channels in the initial reaction space are predicted to be opened at the peak endplate current. The cooperative opening of ion channels by acetylcholine requires that acetylcholine be introduced into the cleft in discrete, concentrated elements. Virtually all the open channels are confined to the initial reaction space, although acetylcholine-bound receptor sites can be much more widely distributed. | US |
This paper develops techniques for equivalent circuit analysis of tight epithelia by alternating-current impedance measurements, and tests these techniques on rabbit urinary bladder. Our approach consists of measuring transepithelial impedance, also measuring the DC voltage-divider ratio with a microelectrode, and extracting values of circuit parameters by computer fit of the data to an equivalent circuit model. We show that the commonly used equivalent circuit models of epithelia give significant misfits to the impedance data, because these models (so-called "lumped models") improperly represent the distributed resistors associated with long and narrow spaces such as lateral intercellular spaces (LIS). We develop a new "distributed model" of an epithelium to take account of these structures and thereby obtain much better fits to the data. The extracted parameters include the resistance and capacitance of the apical and basolateral cell membranes, the series resistance, and the ratio of the cross-sectional area to the length of the LIS. The capacitance values yield estimates of real area of the apical and basolateral membranes. Thus, impedance analysis can yield morphological information (configuration of the LIS, and real membrane areas) about a living tissue, independently of electron microscopy. The effects of transport-modifying agents such as amiloride and nystatin can be related to their effects on particular circuit elements by extracting parameter values from impedance runs before and during application of the agent. Calculated parameter values have been validated by independent electrophysiological and morphological measurements. | US |
The NH exchange rates in aqueous media of oxytocin and 8-lysine vasopressin (LVP) have been measured by using transfer of solvent saturation method. The data are consistent with a "highly motile" dynamic equilibrium between folded and highly solvated conformations. The highly-motility limit applies to the exchange of NH hydrogens of oxytocin and LVP. Folded structures are more prevalent in oxytocin than in LVP. Partial shielding is indicated for peptide hydrogens of Asn5 and perhaps also Cys6 of oxytocin and for Cys6 of LVP. It is tentatively proposed that the folded conformation of oxytocin in aqueous media may contain a parallel beta-structure in the tocinamide ring consisting of two hydrogen bonds: one between the Tyr2 C = O and Asn5 peptide NH as originally proposed for the preferred conformation of oxytocin in dimethyl sulfoxide (D. W. Urry and R. Walter), and the second between he Cys1 C = O and the Cys6 NH. In LVP the hydrogen bond between the Tyr2 C = O and Asn5 peptide NH appears to be absent. The acylic tripeptide sequences (-Pro-X-Gly-NH2) of both hormones appear to be predominantly solvated. The second-order rate constants for acid catalyzed exchange of the primary amide hydrogens of Gln4, Asn5, and Gly9 of oxytocin are consistently greater for the trans NH than for the corresponding cis NH. This observation can be rationalized in terms of mechanisms involving protonation of either the amide oxygen, or the amide nitrogen, but with limited rotation about the C - N bond. | US |
An automated computer prediction of the chain reversal regions of globular proteins is described herein using bend frequencies and beta-turn conformational parameters (Pt) determined from 408 beta-turns in 29 proteins calculated from x-ray atomic coordinates. The probability of bend occurrence at residue i is pt = fi X fi+1 X fi+2 X fi+3 with the average bend probability less than Pt greater than = 0.55 X 10(-4). Tetrapeptides with pt greater than 0.75 X 10(-4) ( approximately to 1.5 X less than pt greater than) as well as less than Pt greater than 1.00 and less than Pa greater than less than less than Pt greater than greater than less than P beta greater than are selected by the computer as probable bends. Adjacent probable bends (i.e., 11-14, 12-15, 13-16) are compared pairwise by the computer, and the tetrapeptide with the higher pt value is predicted as a beta-turn. The percentage of bend and nonbend residues predicted correctly for 29 proteins by this computer algorithm is %t+nt = 70%, whereas 78% of the beta-turns were localized correctly within +/- 2 residues. The average beta-turn content in the 29 proteins is 32%, with helical proteins having fewer bends (17%) than beta-sheet proteins (41%). Three proteins having iron-sulfur clusters were found with the highest percentages of beta-turns: Chromatium high potential iron protein (65%), ferredoxin (57%), and rubredoxin (65%). Finally, the bend frequencies at all 12 positions from 457 beta-turns in 29 proteins (Chou and Fasman, 1977) were used to test the effectiveness of predicting bends using 2, 4, 8, and 12 residues as well as different cut-off pt values. The computer analysis showed that 1.25 less than pt greater than to be the best cut-off yielding 70% accuracy in %t+nt for 4 residues and %t+nt = 73% for 12 residues in predicting the bend and nonbend regions of proteins. | US |
Using the bend frequencies based on 29 proteins in the previous paper (Chou and Fasman, 1979), beta-turn probability profiles were calculated for the C-peptides of 10 mammalian proinsulins, for 7 proteinase inhibitors, and for 12 species of pancreatic ribonucleases. Beta-turn correlation coefficient matrix tables were also computed to obtain the statistical mean between 45 pairs of proinsulin C-peptides, less than Ct greater than = 0.57 +/- 0.31; 21 pairs of proteinase inhibitors, less than Ct greater than = 0.73 +/- 0.13; and 66 pairs of ribonucleases, less than Ct greater than = 0.83 +/- 0.08. Despite relatively low sequence conservation in these three sets of proteins, beta-turns were predicted to be highly conserved: 33% sequence vs. 78% bend for the proinsulins, 20% sequence vs. 85% bend for the proteinase inhibitors, and 65% sequence vs. 92% bend for the ribonucleases. These results suggest that chain reversal regions play an essential role in keeping the active structural domains in hormones and enzymes intact for their specific biological function. | US |
It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by suppression of TPhB- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decreases being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25 - 30% of the total 2,4-D-induced change of the potential difference is between the plane of absorption of TPhB- and the aqueous solution, and the remaining fraction is between the membrane interior and the absorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studied of transport of positive and negative ions across bilayer membranes. In contract, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membranes, but rather with a layer of 2,4-D molecules absorbed at the nonpolar/polar membrane boundary. | US |
The birefringence of frog retinal rod outer segments is analyzed in terms of a three-dielectric layer model. The possibility that the birefringence gradient found in such cells is due to changes in the disk membrane-pair spacing is investigated using previously published glycerol imbibition data (Kaplan et al., 1978. Biophys. J. 23: 59-70). The higher net birefringence of the basal end compared to the midpoint of rod outer segments can be accounted for by a smaller negative form birefringence term due to either a smaller or larger intradiskal space, depending upon the assumed relative solids contents of the intradiskal and cytoplasmic spaces. | US |
Electrochemical properties of Na+-selective glass microelectrodes were studied and compared with those of K+-selective glass microelectrodes. The selectivity of Na+-selective glass microelectrodes depended on the ion concentration of test solutions. With aging, resistance of Na+-selective microelectrodes increased and their selectivity for Na over K decreased. Na+-selective microelectrodes potential measured in NaCl solution remained constant with aging, while the potential measured in KCl solution decreased and became more positive. The changes in resistance and potential of Na+-selective microelectrodes may be due to the effects of the less mobile cation, i.e., H+ or K+ on the Na ion exchange in the Na-sensing region. The results indicate that Na+-selective microelectrodes must be used as soon after filling as possible. The selectivity of Na+-selective microelectrodes increased with increase of the sensitive exposed-tip length, whereas their response time became slow due to a large recessed volume, indicating requirement of an optimum exposed-tip length for intracellular applications. The changes in the properties of Na+-selective glass microelectrodes with aging contrasted with those of K+-selective glass microelectrodes in which resistance decreased and K+-selectivity increased. The K+-selective microelectrodes required aging before use for a high selectivity and low resistance. The K+-selective microelectrodes with low resistance after sufficient aging can be used without insulation to measure K+ and Na+ activities in aqueous solutions. The different properties between Na+- and K+-selective microelectrodes are understandable, because hydration of N+-selective glass is much less extensive than that of K+-selective glass. | US |
A differential equation has been derived for the motion of the mechanosensory hairs of animals when they are stimulated by the motion of their fluid environment. Specific solutions of the equation are obtained for three states of fluid flow including steady-state sinusoidal oscillations. The model is specifically applied to crayfish sensilla in an aqueous medium, but the assumptions of the model are also shown to be valid in air for the sensory hairs of insects. The calculations are consistent with available experimental data. | US |
The possibility is suggested that cardiac aneurysms are formed when an infarcted region of the ventricular wall becomes elastically unstable and "blows out". The consequence of such a blowout could be a large saccular aneurysm or even cardiac rupture. We use a nonlinear stress-strain relation capable of describing both the passive and active myocardial wall to examine this possibility in terms of large-deformation membrane theory. Ventricular infarcts made of a material having physical properties like rubber would be expected to blow out, but those made of passive myocardium would not. | US |
Voltage transients are induced by brief light flashed on bilayer membranes with absorbed 3,3'-bis(alpha-(trimethylammonium)methyl)azobenzene (Bis-Q). The voltages are positive for trans-to-cis photo-isomerization, and negative for cis-to-trans photo-isomerization. The risetimes in phosphatidylethanolamine-decane bilayer membranes indicate that absorbed trans-Bis-Q is photo-isomerized to cis within 2 microseconds, and that cis is photo-isomerized to trans within 15 microseconds. | US |
We have explored the kinetic implications of a model that may account for the acceleration of tail fiber (F) attachment to baseplates (B) by whiskers (W) on bacteriophage T4. The model assumes that a W-F complex is formed initially, and that the tethered fiber then undergoes rotational diffusion until a B-F encounter takes place. In the absence of whiskers, B-F complexes must form unassisted. Formation of a W-F intermediate will accelerate F attachment to B if (a) the bimolecular rate constant for W-F complex formation is larger than that for direct B-F interaction and (b) subsequent rotational diffusion of the tip of F to B is not much slower than the dissociation of W-F. Condition a was investigated by applying a recent theory of orientational effects on translational diffusion-controlled reactions. This theory suggests that substantial rate enhancement is expected if the reaction half-angle theta 0 is larger for W-F than for B-F complex formation. Condition b was investigated by calculating the mean and the variance of the time required for the diffusion of a molecule (the proximal tip of the fiber) on a spherical surface (whose radius is the distance from the tip to the whisker tethering point) into a circular sink (the baseplate site). The mean time is on the order of the inverse rotational diffusion coefficient, DR, of the fiber, but is sensitive to theta 0. Both conditions are satisfied for plausible choices of parameters. The solution to the diffusion equation we have obtained should have application to other physical situations, such as the rate of quenching of a fluorophore as it diffuses on the surface of a spherical membrane into proximity with a quencher. | US |
The permeation of acetic acid through large unilamellar phospholipid vesicle membranes has been investigated using the unique capability of nuclear magnetic resonance to characterize flow under pseudo-equilibrium conditions. Two types of experiments have been employed: total line shape analysis and selective population transfer. These techniques are sensitive to permeation on time scales ranging form 0.001 to 10.0 s. The permeation rate dependence on pH and acetic acid concentration indicates that the neutral acetic acid monomer is the dominant permeant species with a permeation coefficient of 5 +/- 2 x 10-4 cm/s. Mechanisms of permeation and the applicability of nuclear magnetic resonance methodology are discussed. | US |
A model for left ventricular diastolic mechanics is formulated that takes into account noneligible wall thickness, incompressibility, finite deformation, nonlinear elastic effects, and the known fiber architecture of the ventricular wall. The model consists of a hollow cylindrical mass of muscle bound between two plates of negligible mass. The wall contains fiber elements that follow a helical course and carry only axial tension. The fiber angle (i.e., helical pitch) is constant along the length of each fiber but varies through the wall in accordance with the known distribution of fiber orientations in the canine left ventricle. To simplify the analysis and reduce the number of degrees of freedom, the anatomic distribution of fiber orientations is divided into a clockwise and counterclockwise system. The reference configuration for the model corresponds to a state in which, by hypothesis, the transmural pressure gradient is zero, the tension is zero for all fibers across the wall, and all fibers are assumed to have a sarcomere length of 1.9 micrometer. This choice of reference configuration is based on the empirical evidence that canine ventricles, fixed in a state of zero transmural pressure gradient and dissected, demonstrate sarcomere lengths between 1.9 and 2.0 micrometer in inner, middle, and outer wall layers, while isolated ventricular muscle bundles are observed to have zero resting tension when the sarcomere length ranges from 1.9 to 2.0 micrometer. An equation representing the global condition for equilibrium is derived and solved numerically. It is found that the model's pressure-volume relation is representative of diastolic filling in vivo over a wide range of filling pressures, and the calculated midwall sarcomere lengths in the model compare favorably with published experimental data. Subendocardial fibers are stretched beyond Lmax even at low filling pressures, i.e., 5 mm Hg, while fibers located between 60-80% of wall thickness extend minimally between 5 and 12 mm Hg. The hydrostatic pressure field within the wall is highly nonlinear. The pressure rises steeply in the subendocardial layers so that the net gain in pressure in the inner third of the wall is 85% of the filling pressure. It is demonstrated that these results are independent of heart size for a family of heart models that are scale models of each other. They are, however, critically dependent on the existence of longitudinally oriented fibers in the endocardial and epicardial regions of heart wall. | US |
We display the displacement vectors or eigenvectors of calculations of the A- and B-DNA backbones. These calculations are based on a refinement scheme that simultaneously fit several backbone modes of A-DNA, B-DNA, and A-RNA. We discuss the role of symmetry operations in mode calculations and the relevance of these displacement vectors to the interpretation of linear dichroism measurements performed on the A- and B-DNA helix. | US |
Experimental determinations were made of cell number as a function of time for two strains of L5178Y mammalian cells maintained continuously in various environments of radiation. One strain possessed a shoulder in its dose response curve whereas the other did not. Neither strain showed any significant difference in growth rate for interdivision doses on the order of the median lethal dose or less delivered continuously at a low dose rate or pulsed every 4 h at a high instantaneous dose rate. It was also shown that large numbers of dead cells have little effect on growth rate and that these dead cells last as discrete entities for many days. A simple theory of growth rate in the presence of radiation is presented, and the agreement with the observations implies that there is no effect of any sublethal low dose rate radiation received in one generation on the growth rate or radiation sensitivity of the succeeding generation. Further analysis of the data also showed that for the no-shoulder cells at 37 degrees C, tritiated water had a relative biological effect close to unity for cell sterilization. | US |
Molecules present in the serum and ascites fluids of low IgE responder mice previously inoculated with complete Freund's adjuvant have been analyzed in terms of certain biochemical and immunological characteristics. These studies demonstrate that the active molecules, termed "suppressive factors of allergy" (SFA), are (1) nondialyzable, (2) not associated with low-density or high-density lipoproteins, (3) heat stable, (4) precipitable by ammonium sulfate, and (5) approximately 150,000 daltons in molecular size. Studies with immunoadsorbents prepared from various antisera indicate that the suppressive molecules are (1) not immunoglobulin in nature, (2) not reactive with specific anti-H-2 alloantibodies, but (3) reactive with anti-beta 2m antibodies as well as (4) heterologous antisera raised against CFA-immune mouse serum. | US |
Treatment of young mice with lethal X-irradiation (900 R), cyclophosphamide, or hydrocortisone significantly depressed their spontaneous NK activity. The same treatments, however, did not inhibit the augmentation of NK function by poly I:C, suggesting the existence of a treatment resistant pre-NK cell. Both the spontaneous activity and its augmentation were readily inhibited by macrophage-toxic agents such as silica and carrageenan in vivo. Since the NK cells themselves were not macrophages, as shown by the inability of silica or carrageenan to block in vitro cytolysis of target cells, we postulated that macrophages were required to maintain NK activity in vivo and that they were essential accessory cells in the augmentation. The induction of interferon by poly I:C was also inhibited by silica and carrageenan. The augmentation of NK activity induced by poly I:C was consistently accompanied by the rise in serum IF levels of the treated mice, and its inhibition by the macrophage-toxic agents was followed by decreased production of interferon. These observations support our hypothesis that macrophages, in response to poly I:C, produced interferon which in turn activated NK cells to become cytolytic. | US |
The effect of increased stimulus level on critical bandwidth was investigated. Noise-masked, pulsed-tone thresholds were obtained by a Bekesy tracking procedure from 4 practiced normal-hearing adults at .5, 1, 2, and 4 kc/s. Tones were placed symmetrically within a band-reject region. Critical bandwidth was taken as the frequency separation between noise bands at which masked tonal threshold began to change and was found to increase fairly regularly as the spectrum level of the noise increased from 30-60 db SPL. However, the data at 60 db SPL may have been influenced a 2 and 4 kc/s by the detection of aural distortion products. The suggestion was made that when signal frequency is outside the spectral limits of the masker, critical bandwidth widens as masker level is raised; however, when signal frequency is within the spectral limits of the masker (as with a continuous noise wit no notches), critical bandwidth remains unchanged up to 80 bd SPL. | US |
Platelet structural physiology has contributed significantly to our understanding of basic mechanisms of platelet function in hemostasis and thrombosis. Current evidence indicates that platelets are a form of muscle cell with specialized capabilities for secretion and adhesion-aggregation. Activation of the discoid cell by any agent appears to involve a perturbation of the membrane resulting in movement of calcium from the cell wall to the interior. The calcium flux stimulates phospholipase A2 to cleave arachidonic acid from platelet phospholipids starting the cascade of prostaglandin synthesis. In addition, the movement of calcium to the cytoplasm initiates contraction leading to shape change. Products formed during prostaglandin synthesis, particularly thromboxane A2, act as ionophores to transport additional calcium from the dense tubular system to the cytoplasm amplifying the wave of contraction. Alterations in organelle membranes result in their fusion with channels of the open canalicular system. The contractile wave causes extrusion of secretory products which stimulate other platelets to become involved in formation of irreversible aggregates in vitro and hemostatic plugs in vivo. Mechanisms regulating platelet stimulation-contraction-secretion coupling are currently under investigation. | US |
We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV. | US |
An analytic solution of the Förster energy transfer problem in two dimensions is presented for the case in which the orientation factor is independent of the donor-acceptor distance, and both the donors and acceptors are randomly distributed in a plane. A general solution based on the method of Förster is possible since all distances are measured in units of R0. The analytic solution is extended to the cases of donors embedded in structures that exclude acceptors, and donors that bind acceptors. The validity of the analytic solutions is demonstrated by comparison with numerical simulation calculations. Numerical approximations to the exact solutions are given for ease of computation. Specific applications to the case of fluorescence quenching of a membrane-bound donor by membrane-bound acceptors are presented. | US |
We have measured the force-velocity curves of glycerinated rabbit psoas fibers over a range of ATP concentration from 2.5 microM to 5 mM. As the ATP concentration is increased, the isometric tension increases to a maximum around 50 microM, then decreases to a plateau at 70% of the maximum by 1 mM ATP. At low ATP concentrations the maximum velocity of contraction is low and increases with increasing ATP, reaching a plateau at approximately 2 lengths per second by 1 mM ATP. Our studies suggest that the binding of ATP dissociates the myosin head from actin in the contracting muscle, a reaction similar to that seen in solution. We have constructed models of the actin-myosin-nucleotide interactions based on a kinetic scheme derived from solution studies. The fit of these models to the data shows that the rates of some reactions in the fiber must be considerably different from the rates of the analogous reactions in solution. The data is best fit by models in which head attachment occurs rapidly at the beginning of a power stroke, head detachment occurs rapidly at the end of the power stroke, and the force produced by a myosin head in a power stroke is independent of velocity. | US |
The neutral anesthetics chloroform and benzyl alcohol, at concentrations that block the nerve impulse, greatly modify the transport parameters of positive and negative ions in lipid bilayers made from monolayers. Both chloroform and benzyl alcohol increase the membrane permeability to these ions and increase the translocation rate for tetraphenylborate. It was found that both anesthetics increase the membrane permeability to positive ions more markedly than to negative ions. It was also found that the membrane capacitance increases lineary with the concentration of benzyl alcohol. At 51 mM benzyl alcohol, the increase in capacitance is approximately 6%. Chloroform also increases the membrane capacitance; the increase in capacitance was found to be 6% at 18 mM chloroform. An analysis of the changes in the transport parameters of the lipophilic ions, together with the changes in membrane capacitance, suggests that benzyl alcohol and chloroform modify the dipole potential and dielectric constant of the membrane. Benzyl alcohol may also increase the "fluidity" of the lipid bilayer membranes. At 36 mM benzyl alcohol, the membrane permeability to acetamide increases by 38%. | US |
A theoretical formulation and experimental methodology are presented for a new multipoint analysis of membrane translational dynamics. The redistribution of fluorescent probe after a localized photobleaching pulse is monitored at several locations by a focused laser beam sequentially scanned through the bleached area. The spatial information so obtained provides a unique sensitivity to possible systematic flow and a direct internal calibration of the characteristic transport distance. These capabilities are demonstrated with experimental data on a reconstituted multibilayer system. | US |
The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis. | US |
An improved method for internally perfusing the Myxicola giant axon based on removing the axoplasm by dispersing it in KCl-KF salt solutions is described. Proteolytic enzymes are not introduced. With this improved method perfused preparations show long-term stability of their electrical properties and the ability to generate action potentials for many hours. Mean initial values for resting membrane potential, action potential amplitude, and peak inward current were -68 mV, 118 mV, and 3.62 mA/cm2, respectively. Mean resting membrane resistance was 75% of that in intact axons. In one series of voltage clamp experiments, perfused preparations remained excitable for a mean period of 5 1/2 h, but this period could exceed 10 h. 4 min are needed for exchange of internal solutions. At least 50 mM KF is required both in the axoplasm liquefying solution and in the standard perfusate to obtain stable preparations. | US |
Small clusters of ventricular cells prepared from 7-d chick heart maintain spontaneous, stationary, rhythmic beating in culture for many hours. For clusters containing I-125 cells, mean interbeat interval (IBI) is 0.45 +/- 0.08 s and is independent of cell number (N), whereas, the coefficient of variation of IBI (C) is proportional to N-1/2. Because membrane voltage noise in such clusters would also be expected to vary as N-1/2, we propose a model relating fluctuation in IBI (sigma IBI) to voltage noise (sigma v). A simplified model consisting of random voltage fluctuations superimposed upon a linear pacemaker depolarization of slope a is used to analyze the N-dependent shape of the IBI histogram. Values of sigma v derived from the relation sigma IBI = sigma v/a, or calculated from the skewness of the measured IBI histograms, both agree well with those extrapolated from steady-state noise recorded from resting heart-cell aggregates. | US |
The effect an abrupt boundary has upon the dynamical response of a neural network is investigated. The retina of the Limulus eye is used as a model system for studying this effect. A theoretical technique is presented for the quantitative prediction of the manner in which this neural network responds in the vicinity of its boundary. Corresponding experimental measurements of the response to moving stimuli by single optic neurons located near retinal boundaries are presented. Theory and experiment show detailed quantitative agreement. | US |
The interactions between vitamin-A and beta-lactoglobulin have been investigated. We have found that two different complexes can be formed: one involving vitamin-A, and one involving a derivative of vitamin-A that most probably has a retro-beta-ionylidene structure. Room temperature absorption spectra for these complexes in phosphate buffer at pH 7.50 are reported and discussed. | US |
The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4. | US |
The autocorrelation function of a given process is related to its spectral density by the Wiener-Khintchine theorem, and both expressions contain the same information. We report here a measurement of the current noise produced in a lipid bilayer membrane doped with hydrophobic anions of dipicrylamine. The results are in good agreement both with relaxation measurements on the same membrane and with an analysis of the spectral density of the current noise for this system which has been presented by other workers. Although measurement of the spectral density function is generally more complete for technical reasons, the autocorrelation function provides, for the case studied here, more physical insight into the underlying charge transport mechanism. We find that the measured autocorrelation function is negative at short, but nonzero, times. This is a consequence of the operative conductance mechanism in this case, which cannot carry current continuously in the same direction without compensatory reverse flow. | US |
Incubations of adult rat uterine tissue and human endometrial and myometrial homogenates with [1,2,6,7-3H]testosterone resulted in the formation of 5 alpha-[3H]dihydrotestosterone. The rat uterine homogenates showed higher activity than the minced tissue. The activity in human tissue was found to be less than rat tissue on a per unit weight basis. The human endometrium has been found to possess significantly higher activity than myometrium. These results support the hypothesis that androgens may exert their effect on uterus via 5 alpha-reduced metabolites. | US |
Bile acid sequestrant resins can bind T4 in vitro and in the gut. Therefore, the effect of colestipol HCl on thyroid function was studied prospectively in 17 subjects with type II hyperlipoproteinemia. Serum T4, T3, T3 resin uptake, and TSH levels were measured before and during therapy. No change in T4 levels and only small transient, but significant, decreases in T3 were noted in a minority of patients; TSH levels did not change. Small increases in T3 resin uptake were noted. We conclude that no major alterations in thyroid function occurred with colestipol therapy. | US |
Peritonitis in rats was produced by cecal ligation and puncture. Sixteen hours following cecal ligation and puncture, the gangrenous cecum was removed and the animals received either 4 ml saline (nontreated), 0.75 ATP-MgCl2 (100 mumoles ATP plus 50 mumoles MgCl2), and 2.0 ml of 50% glucose or 2.0 ml of 50% mannitol and 1.25 ml saline. Two hours after the removal of the cecum, RES function was evaluated by measuring the intravascular clearance of a 131 I triolein-labeled gelatinized test lipid emulsion. The intravascular half-time (t1/2) in the nontreated animals was double that of sham-operated animals, suggesting that significant depression in RES function occurred during sepsis. Administration of ATP-MgCl2 plus glucose following sepsis resulted in t1/2 values similar to those of sham-operated animals, indicating that the impairment of pagocytic activity of the RES was reversed with treatment. The beneficial effect of treatment following sepsis does not appear to be due to hypertonicity, since administration of 50% mannitol failed to decrease the t1/2. The precise mechanism of the beneficial effect of ATP-MgCl2 + glucose on restoration of RES function is not known. | US |
This study evaluates the effects of 30 mg/kg methylprednisolone (MP) administered to eight trauma patient over a 30-minute period during initial resuscitation. Vascular pressures, cardiac index (CI), left ventricular stroke work index (LVSWI), systemic (SVR) and pulmonary (PVR) vascular resistance, oxygen delivery (OD), oxygen consumption (VO2), physiological shunt (shunt), limb blood flow (LBF), limb-oxygen delivery (LOD), and limb oxygen consumption (VLO2) were calculated at control and 1, 2, 4 and 6 hours following MP administration. At 1 hour there was an increase in CI from 2.9 +/- 0.3 to 3.5 +/- 0.3 liters/min/m2 (P less than 0.01), in OD from 961 +/- 172 to 11067 +/- 148 ml/min (P less than 0.05), in VO2 from 178 +/- 16 to 220 +/- 16 ml/min (P less than 0.01), in shunt from 25 +/- 3% to 33 +/- 3% (P less than 0.05), and a decrease in SVR from 1187 +/- 98 to 1945 +/- 87, and in PVR form 222 +/- 22 to 178 +/- 18 dyne sec/cm5 (P less than 0.05). These values returned to control by 4 hours. In spite of a pulmonary wedge pressure (PWP) that did not increase form a control of 5 +/- 2 mm Hg, and a mean arterial blood pressure (MABP) that did not decrease from a control of 86 +/- 5 mm Hg, LVSWI increased significantly at 1 hour (P less than 0.01). LBF, LOD, and VLO2 decreased at 1 hour (P less than 0.05). Since increased cardiac output was associated with increased stroke volume and left ventricular stroke work index, but without an increase in preload (PWP) or a decrease in afterload (MABP), methylprednisolone(MP), in pharmacologic dosage appears to have a positive inotropic effect on the myocardium of trauma patients during resuscitation. | US |
Numerous studies have demonstrated that reticuloendothelial system (RES) depression induced by colloid blockade increases susceptibility to circulatory shock following trauma and sepsis. Recent data have suggested that this may relate to the failure of the RES to clear potentially embolic material derived from activation of the hemostatic system. The present study thus compared the hypotensive response precipitated by trauma or sepsis with that resulting from induction of intravascular coagulation. Mean arterial blood pressure (MABP) was monitored for 120 minutes after sublethal NCD trauma and after intra-aortic injection of live E coli (approximately 10(10) organisms per rat), E coli endotoxin (0.1 mg/100 gm), or bovine thrombin (10 units/100 gm) in 400-500 gm rats 30 minutes after RE blockade (50 mg/100 gm gelatinized lipid colloid) or saline injection. All rats were anesthetized with sodium pentobarbital. No hypotension was observed in blockaded control rats. After trauma, MABP decreased by 20 minutes after injury and recovered to normal levels by 1 hour post-trauma. MABP decreased in blockaded rats after trauma and remained diminished through 2 hours. After live E coli endotoxin or thrombin, both the normal and the blockaded groups underwent an initial hypotension of similar magnitude. A second period of hypotension was much more pronounced in the RE-blockaded animals. Reduced MABP persisted in these animals through 2 hours. These data indicate that RE blockade enhances the hypotensive response to intravascular coagulation and that resulting from trauma or sepsis. This effect was especially apparent during the second phase of hypotension during sepsis and intravascular coagulation. It was suggested that the RES manifests some protective effect against the agents inducing this secondary hypotensive response. | US |
Reticuloendothelial system (RES) depression has been correlated with diminished resistance to trauma, shock, and sepsis in man and animals. Previous studies have related the depression of RES hepatic Kupffer cell phagocytic function after trauma to diminished bioassayable opsonic activity. The present study determined if the loss of biological activity and RES alteration correlated with immunoreactive serum opsonic alpha 2 SB glycoprotein levels after trauma. Serum opsonic activity was measured by liver slice bioassay, and immunoreactive opsonic protein was measured by rocket electroimmunoassay. RE function was determined by colloid clearance over a 24-hour post-trauma period. Anesthetized rats (250-300 gm) subjected to sublethal or severe (greater than LD50) whole-body NCD trauma were the shock models investigated. Immunoreactive levels in 63 rats prior to injury were 518 +/- 24 microgram/ml. Neither biological nor immunoreactive levels were altered over 24 hours in anesthetized sham-traumatized controls. Temporal alteration in the initial decrease and recovery pattern of biologically active and immunoreactive opsonic protein levels significantly correlated following both sublethal and severe injury. Moreover, the patterns of immunoreactive levels of the opsonic protein correlated with the functional phagocytic activity of the RES as determined by vascular clearance of a test dose of blood-borne radiolabeled particulates. This glycoprotein falls after trauma, and the magnitude and duration of the decline increases with severity of injury. Immunoreactive opsonic alpha 2 SB glycoprotein appears to be an accurate measurement of circulating opsonic activity and RE Kupffer cell function after trauma, especially with respect to clearance. Thus, immunoreactive opsonic protein warrants clinical consideration as a noninvasive measure of reticuloendothelial systemic defense in patients after trauma and burn. | US |
To study the effect of interstitial edema on tissue oxygen transport, we infused buffered isotonic saline solution at a rate of 200 ml/kg/hr into mongrel dogs for two hours, measuring oxygen delivery and consumption in a skinned, innervated hindlimb. Hematocrit dropped gradually to 10.8 +/- 3.9%. Muscle water content increased 13% (P less than 0.05). However, oxygen consumption did not change significantly from control of 0.228 +/- 0.029 ml/min/100 gm. Femoral venous oxygen tension fell from control of 62.6 +/- 2.6 mm Hg to 31.4 +/- 2.3 mm Hg. Mean arterial flow increased to nearly twice the control level of 12.5 +/- 1.1 ml/min/100 gm and fell gradually, with the sustained 50% drop in vascular resistance largely explained by expected decreases in blood viscosity. We conclude that interstitial edema did not cause a significant defect in canine skeletal muscle oxygen utilization. | US |
A visual search task was administered to eighteen neurosurgical patients and fourteen control subjects on four occasions over a period of six months. Improvement occurred during the first five postoperative weeks but there was evidence of persistent impairment when patient performance was compared to control performance - the comparison makes use of a simple mathematical model. | US |
Surgical advances often call for close radiologic scrutiny to assess their success or complications. At the same time, diagnostic radiology is also constantly developing new approaches. It is for this reason that we describe the radiographic analysis of several current surgical and radiological procedures which should be of interest to the practicing clinician. The current surgical procedures considered are gastric bypass for morbid obesity and continent ileostomy. The current new radiologic procedures include techniques for performing small bowel enema, per-oral ileal and colonic examinations with retrograde air insufflation, and improved evaluation of the postantrectomy stomach using double-contrast techniques. | US |
In 1970, it was found that sera removed from rats 24 hr after unilateral nephrectomy stimulate the incorporation of 3H-Thymidine into the DNA of incubating rat renal cortex. The same sera did not influence isotope incorporation into the DNA of incubating liver, spleen, and lung tissue. Later, a substance was described in renal tissue that enhanced the action of the renotropic factor in sera. It was proposed that this tissue factor activated the circulating renotropic substance and that lack of this activator in other tissues was responsible for the specificity of the circulating renotropic substance to renal tissue. The present study suggests that this is the case as the addition of the renal tissue factor and sera from uninephrectomized rats can stimulate 3H-Thymidine incorporation into the DNA of rat liver slices. | US |
Bone marrow granulocyte progenitor cells (CFU-C) were assayed in methyl-cellulose prior to cryopreservation in Dimethylsulfoxide (DMSO) and after thawing and diluting the DMSO. The time of dilution from 10% to 1% DMSO and the temperature of the sample and diluting media were studied. Compared with samples diluted at 0 degrees-4 degrees C, samples which were diluted at 24 degrees C were more viable by Trypan Blue exclusion (p less than .01) and had greater CFU-C growth in vitro (p less than .01). There was no advantage to prolonging dilution time from 10 minutes at a constant rate to 40 minutes using stepwise technique. Recovery of CFU-C at 24 degrees C ranged from 40% to 114% with a mean +/- S.D. of 67% +/- 19.5%. There was evidence that clonogenic cells were selectively preserved under the conditions described. | US |
Pyran Copolymer, divinyl-ether maleic anhydride increases the concentration of circulating pluripotential stem cells in mice by a factor of 15 to 30. Maximal mobilization occurs five days after Pyran Copolymer injection with synchronous peaks of CFU-S and CFU-C. When Pyran fractions of defined molecular weight from 12,000 to 52,000 are injected into mice, mobilization of CFU-S and CFU-C parallels molecular weight. Hemopoietic stem cells are mobilized from bone marrow into peripheral blood and subsequently trapped in the spleen. | US |
The continuous in vitro marrow culture system for proliferation of mouse pluripotent hemopoietic stem cells (CFUs) and granulocyte-macrophage progenitor cells (CFUc) as initially described by T. M. Dexter, depended upon a 25% concentration of special lots of horse serum and addition of fresh "recharging" marrow after 3-4 weeks. This system has been modified to permit longer hemopoiesis in non-recharged cultures over 25-30 weeks. Addition of 10(-7)M hydrocortisone sodium hemi-succinate during weekly feeding and switch from horse to 25% fetal calf serum with corticosteroid at week 4, maintains stability of the adherent marrow stromal cells, decreases lipogenesis (which is deleterious after 10 weeks) and increases proliferation of hemopoietic cells over longer duration. Stem cells removed from 8 week old primary NIH/Swiss marrow cultures reconstituted hemopoiesis in lethally irradiated mice. While 16 week old cultures produced total numbers of CFUc equivalent to 8 week cultures, these cells could not prevent marrow death following similar total body irradiation. Thus, stem cells moved progressively from a pluripotent to a committed CFUc compartment between weeks 8-16. Human femur marrow required thyroxine in addition to hydrocortisone for sustained CFUc maintenance over a shorter 8 week period. Improved technology for mouse long-term marrow cultures should further aid in developing a usable human "Dexter-system". | US |
A number of assays exist for hematopoietic stem cells in both humans and mice, but the appropriate stem cell assay for the repopulating potential of human marrow is not clear. Two murine models suggest that these assays may not always predict marrow proliferative potential. In vivo diffusion chamber culture growth of CD1 marrow depleted of pluripotent stem cells (CFU-S) by exposure to mouse-brain antisera plus complement was equivalent to or greater than that of normal serum treated control marrow. Furthermore, CF1 mice repeatedly injected with endotoxin had markedly stimulated granulopoiesis with increases in the number of marrow CFU-S and the % in S phase but no changes in the number or proliferative status of marrow CFU-C. However, inbred BDF1 mice chronically injected with endotoxin although also showing striking increases in granulopoiesis had no significant alteration in their marrow CFU-S or CFU-C number or cell cycle status relative to saline injected controls. Both models present examples where conventional stem cell assays do not provide insight into marrow cell production and suggest that in vitro clonal assays of human marrow cells may not always predict for the potential of marrow to repopulate a human transplant recipient. | US |
This study was designed to investigate quantitatively the interference of thyroglobulin autoantibodies in the RIA of human thyroglobulin (hTG). Anti-hTG autoantibodies were combined with purified hTG to produce samples with known antibody titers and hTG concentrations. These samples were analyzed in the RIA. By using anti-human globulin serum it was first shown that immune complexes formed between labeled hTG and human anti-hTG. It was then shown that the most important factor in determining the direction of the interference was the specificity of the precipitating (second) antiserum with respect to these immune complexes. When the precipitating antiserum was specific, i.e. did not recognize human antibodies, the immune complexes remained in the supernatant and the measured hTG concentration was falsely elevated. When the precipitating antiserum cross-reacted with human antibodies, the direction of the interference depended on the sample volume. At small volumes there was false depression while at large volumes there was false elevation of apparent hTG levels, depending on the capacity of the precipitating antiserum to combine with human antibodies. Anti-hTG titers far below those detected by the tanned-red cell hemagglutination test had very large effects, to the point where measurements of hTG could not be made, when a cross-reactive precipitating antiserum was used. Therefore, the procedure which investigators have used until now, to exclude samples with anti-hTG hemagglutination titers above an arbitrary limit, is not adequate. It is necessary, until methods are developed which avoid the problem of autoantibody interference, to characterize each assay to determine the limits of anti-hTG that can be tolerated. The factors which influence anti-hTG interference in the hTG RIA are 1) the specificity of the precipitating antiserum, 2) the sample volume, 3) the maximum tracer binding, and 4) the anti-hTG titer. | US |
A sensitive RIA for human calcitonin has been developed which can detect 1-2 pg hormone. This procedure permits the measurement of the low concentrations of calcitonin in the unextracted plasma of normal human subjects. In 55 normal adults, mean plasma calcitonin was 24 pg/ml with an SD of +/- 18 pg/ml, an SE of +/- 2 pg/ml, and a range of less than 10 - 75 pg/ml. There were no discernible age or sex differences in basal hormone concentration. Infusions of calcium, pentagastrin, and glucagon stimulated plasma calcitonin, whereas food and oral calcium did not. The stimulatory effect of pentagastrin was greater in males than in females. These data demonstrate that the low concentration of calcitonin in humans can be stimulated by several secretagogues and suggest that females may have decreased calcitonin reserve. | US |
Haloperidol, a central nervous system dopamine blocker, was given im to seven normal volunteers at a dose level (1.0 mg) known to have central nervous system effects. Plasma PRL levels rose sharply in response to haloperidol, but plasma arginine vasopressin levels did not change significantly. These data do not support the hypothesis of a prominent dopamine neurotransmitter regulation of arginine vasopressin secretion. | US |
Exposure to the extrauterine environment is associated with marked stimulation of the hypothalamic-pituitary-thyroid system after which, pituitary-thyroid equilibrium must be reestablished. This marked endogenous perturbation offers the opportunity to study the manner in which the pituitary-thyroid axis is reequilibrated. T4, T3 and TSH concentrations have been measured by RIA in sera from 440 healthy newborn infants, whose ages ranged from birth to 236 h. Results were analyzed by nonlinear curve-fitting procedures to assess the changes in mean hormone concentrations with age (t). Equations have been derived by Danziger and Elmergreen to allow assessment of oscillatory behavior during hormone equilibration. Applying these equations to the present data, we observed the presence of an oscillatory cosine term in the equation for each hormone. This indicates significant oscillations in serum T4, T3, and TSH concentrations during the first 5 days of life. The period of the oscillations approximates 16 h. The oscillations in T4, lag 1/2 to 3/4 cycle behind TSH; T3 lags behind T4. Thus, disturbances in hypothalamic-pituitary-thyroid equilibrium seem to be followed by periodic oscillations in hormone concentrations; these oscillations decrease in amplitude as the negative feedback system establishes new equilibrium conditions. | US |
Experiments were performed on a particulate fraction from human parathyroid glands. A high activity of adenylate cyclase was detected which was linear with time and protein concentration. The enzyme had an optimum pH in the range of 7-8 and a Km for ATP of 0.44 X 10(-3) M. Ca++ had a profound inhibitory effect; a concentration of 0.5 mM Ca++ reduced enzyme activity by 60%. Maximal enzyme activity was obtained with 5 mM Mg++; higher concentrations of this cation also inhibited enzyme activity. The effect of Mn++ was similar to that of Mg++. Enzyme activity was stimulated by NaF, catecholamines, glucagon, and calcitonin. The effect of catecholamines seems to be mediated through beta-adrenergic receptors. | US |
The present study was designed to compare the immunological, physical, and biological properties of native hCG with an hCG molecule secreted ectopically in vitro by an ovarian adenocarcinoma cell line maintained in long term tissue culture. The hCG produced by the cell line was concentrated by ultrafiltration of the tissue culture medium. The inhibition curves generated by serial dilutions of the culture medium concentrates were parallel to those obtained with purified urinary hCG in the beta-hCG RIA and the rat Leydig cell radioreceptor assay (RRA). The ectopic hCG also reacted with an antibody generated against the carboxyl-terminal peptide (109-145) of beta-hCG. The immunoreactive material cochromatographed with urinary hCG on a Sephadex G-100 column, as determined by the beta-hCG RIA and RRA. Neither free alpha nor free beta subunits were found in the tissue culture medium. The tissue culture gonadotropin was adsorbed onto a Concanavalin A-Sepharose column and could be eluted with alpha-D-methylglucoside. The biological activity of the ectopic hCG was 9289 IU/mg, as determined by the ventral prostate weight (VPW) method in hypophysectomized immature male rats. The biological to immunological ratios by the ventral prostate weight method and RRA were 1.79 and 2.17, respectively. The in vivo disappearance rate of ectopic hCG after injection into immature female rats was significantly faster than that of placental or urinary hCG, but was considerably slower than the disappearance rate of human LH. These studies demonstrate that the immunoreactive and biologically active portions of the hCG produced by the ovarian adenocarcinoma cell line and native hCG are similar or identical. The faster disappearance rate of the ectopic hCG in the rat model may be due to incomplete sialylation of the oligosaccharide moiety of the hCG molecule. | US |
We previously described an RIA for aldosterone based on highly specific antibodies elicited with aldosterone-3,20 dioxime bovine gamma-globulin (Poulsen, K. J. Sancho, and E. Haber, Clin Immunol Immunopathol 2: 373, 1974). We now report the development of higher affinity antibodies of similar specificity that allow the direct measurement of normal aldosterone concentrations in extracts of 100 microliters plasma. The detection limit at +/- 2 SD of mean zero value was 0.2-0.3 pg. The assay was validated by 1) zero values in adrenalectomized plasma, 2) parallel displacement plots of aldosterone standards and plasma extracts, 3) 97.7 +/- 2.5% recovery of added tracer, 4) a correlation coefficient of 0.992 and a slope of 1.08 in a plot of added vs. recovered unlabeled aldosterone, 5) 10.2% interassay variation and 5.6% intraassay variation, and 6)failure of high concentrations of a variety of steroids added to plasma to perturb the measured aldosterone concentration. The simplicity of the assay results in a high degree of productivity, in that 195 plasma samples may be processed in duplicate, in addition to standard curves, in a single working day. | US |
An in vitro system has been developed to examine the effects of PRL on the normal primate mammary gland. alpha-Lactalbumin a milk protein, was found in breast tissue samples from 17 of 19 primates representing several Macaca and Papio species; concentrations ranged from 10-768 ng/mg protein. That none of the animals was pregnant or lactating and half were nulliparous indicates that milk protein production takes place under normal circumstances, even in breast tissue of nonlactating animals. Studies of the effect of PRL on alpha-lactalbumin production in these tissues in organ culture revealed that PRL maintained existing or stimulated new production of alpha-lactalbumin for periods of up to 9 days. Measurement of alpha-lactalbumin in medium bathing mammary tissue from three animals revealed that mean alpha-lactalbumin production during days 7-9 when PRL was added (100 and 1000 ng/ml) was 11 and 59 times greater, respectively, than control. Simultaneous measurement of tissue concentrations of alpha-lactalbumin revealed that those tissues maintained with PRL (1000 ng/ml) had a mean concentration of alpha-lactalbumin that was 61 times that of controls without PRL. PRL consistently maintained or increased alpha-lactalbumin production in tissues from all 22 primates tested. Even in those premenarchal animals in whose mammary tissue alpha-lactalbumin was undetectable initially, PRL stimulated alpha-lactalbumin production in a dose-related fashion. In contrast, when PRL was absent from medium, alpha-lactalbumin concentrations decreased at 9 days to less than 20% of the initial 3-day value in all cases. These studies provide evidence that mammary tissue from normal nonlactating, nonpregnant primates produces milk proteins and that when tissues are exposed to PRL in culture, production of alpha-lactalbumin is stimulated. | US |
Two three-year-old boys with dwarfism (height ages 1-4/2 and 1-11/12 years) and delayed bone ages (1-4/12 and 1-9/12 years) had normal growth hormone (GH) responses after stimulation and low levels of somatomedin. Unlike patients with Laron syndrome, the two patients generated normal levels of somatomedin after administration of exogenous hGH. Treatment with hGH (2 IU every other day) brought about a significant increase in the growth rate of both patients. The growth rate of the first patient increased from 2 cm/year before treatment to 12 cm/year on therapy. The growth rate of the second patient was 4.5 cm/year before treatment, and 8.3 cm/year while on treatment. The two cases represent a new syndrome of dwarfism which may be caused by secretion of a biologically inactive but immunoreactive GH. | US |
T4, T3, rT3, and TSH were measured in cord blood of infants born in mild to severe endemic goiter areas and nonendemic urban areas in South and North America. A significantly high rT3 concentration was found in newborns living in the urban area of South America as compared to a Canadian newborn population. There were no differences among the various thyroid parameters in the groups of newborns studied. Blood from goitrous mothers showed significantly higher concentrations of T3 and TSH than nongoitrous mothers. However, these changes did not affect the concentrations of thyroid hormones, rT3, or TSH in the fetal circulation. | US |
The conversion of radiolabeled androgen to estrone and 17 beta-estradiol was assessed in tissues of human embryos that varied from phenotypically indifferent stages (1-3 cm crownrump length) to midgestation (15. 1-20 cm crownrump length). Significant rates of estrogen synthesis were demonstrated only in ovaries, liver, and brain. Estrogen synthesis was undetectable in gonads from 1-3 cm fetuses, but by the 3.1-5-cm stage it had reached an average rate of 1.9 pmol . h-1 . mg protein -1 in ovaries and remained at this level of activity through the latest stages examined. Estrogen formation was undetectable in testes at all stages examined, but the time of appearance of the capacity to form estrogens in the fetal ovary is similar to the onset of the capacity of the fetal testis to synthesize testosterone. The capacity of the fetal ovary to form estrogen develops before histological differentiation of the tissue. | US |
Hemoglobin A1c concentration (HbA1c) was compared to the plasma glucose responses at 1 and 2 h of an oral glucose tolerance test (OGTT) in 63 subjects preselected because of postprandial hyperglycemia. HbA1c concentrations were correlated with 1- and 2-hour plasma glucose responses during the OGTT (r = 0.776 and 0.8602, respectively). The OGTT responses were diabetic-like in 21, indeterminate in 15, and normal in 27 subjects. HbA1c values were within normal limits in all subjects who had a normal or indeterminate OGTT response and in 10 out of 21 with a diabetic OGTT. The 2-h OGTT response among the 10 diabetic responders with normal HbA1c was 200 +/- 31 mg/100 ml (mean +/- SD), while that of the 11 diabetic responders with elevated HbA1c was 352 +/- 122 mg/100 ml. All subjects with an elevated HbA1c had a 2-h plasma glucose above 228 mg/100 ml, whereas only 7% of subjects with a normal HbA1c had a 2-h glucose above this value. It is concluded that only about half of the patients currently diagnosed as having mild or chemical diabetes by OGTT have elevated HbA1c and that an elevated HbA1c is usually associated with 2-h OGTT levels above 228 mg/100 mg. | US |
This study was performed to assess the relative contributions of the fetal and definitive zones of the human fetal adrenal gland to "corticoid" (cortisol and perhaps other corticosteroids) and dehydroepiandrosterone sulfate (DHAS) production, and the possible regulatory role of ACTH and the fetal pituitary in the secretion of of these steroids. Corticoid and radioimmunoassayable DHAS or total aromatizable androgen secretion by the isolated definitive and fetal zones of the human fetal adrenal gland between 10-20 weeks gestation has been studied in a superfusion system. Different functional capacities of the two zones were seen; corticoids were found to be secreted primarily by the definitive zone, while DHAS was found to be the main secretory product of the fetal zone. Addition of ACTH (250 ng/ml) or fetal pituitary homogenate produced a 2- to 5-fold stimulation of corticoid production by the definitive zone at all gestational ages studied. DHAS secretion by the fetal zone was also stimulated by ACTH. These results indicate that the definitive and fetal zones of the human fetal adrenal gland at midgestation have the capacity to respond to ACTH with increased corticoid or DHAS secretion, respectively. | US |
Calcium metabolism during the menstrual cycle was studied in seven women from whom fasting blood samples were drawn daily or every other day throughout ovulatory cycles. Total calcium (Ca), ionic calcium (Ca++), magnesium (Mg), phosphorus (P), and immunoreactive parathyroid hormone (PTH) and calcitonin (CT) were measured. LH levels were used to date each cycle and progesterone levels were used to confirm ovulation. Plasma estradiol was measured in two of the subjects. In six subjects with cycle lengths of 27-31 days, PTH levels rose progressively through the follicular phase to a peak at or slightly before the LH surge, then fell progressively through the luteal phase; peak PTH levels were 30-35% above early follicular and late luteal values. CT levels were also highest at midcycle, but the CT pattern was somewhat more variable than that of PTH. Ca++ tended to fall until 3-4 days before ovulation and then to increase, while Ca, Mg, and P exhibited no particular pattern. One subject experienced a prolonged (44 day) ovulatory cycle characterized by three distinct PTH peaks, each of which coincided with elevations in plasma estradiol level. These results represent the first report of menstrual cyclicity in calcium-regulating hormones. The timing suggest an estrogen effect and it is hypothesized that estrogen inhibits PTH-induced bone resorption, lowering serum Ca++, which in turn provokes a compensatory PTH output. With the decline of the preovulatory estrogen peak, Ca++ levels rise and PTH secretion falls. Alternatively, it is possible that the primary action may be an estrogen-induced rise in CT release, causing hypocalcemia and consequent PTH output. Cyclic changes in PRL release or vitamin D metabolism might also be involved. | US |
Calcitonin has been considered of therapeutic value in osteoporosis because of its effects in tissue culture. In the whole animal, however, the predominant result seems to be hypocalcemia, which might be expected to have the opposite effect of stimulating parathyroid hormone secretion and therefore resorption of bone. Indeed, in a short term study of 3- and 4-month duration in osteoporotic women, this was found to be so. A combination of calcium and calcitonin was therefore considered a more promising therapeutic alternative for this disease. Calcium was given to 26 patients, alone or with vitamin D, for a period of 15 months, and the effects on serum and urine calcium and phosphorus and on bone resorption and formation were evaluated. Calcium and vitamin D decreased serum parathyroid hormone levels, reduced bone resorption, and increased urinary calcium. The addition of calcitonin to the calcium and vitamin D did not seem to change these effects. Neither form of treatment resulted in change of bone mass. Calcium, with or without vitamin D supplements, may prevent the development of osteoporosis, but it seems unlikely that calcitonin has any additional desirable effect in the disease. | US |
Two siblings with 46,XY male pseudohermapthroditism were demonstrated to have the phenotype characteristic of 5 alpha-reductase deficiency, namely normal testes and male Wolffian duct derivatives (epididymis, vas deferens, and seminal vesicle) terminating in a blind-ending vagina. Clitoromegaly was present at birth and increased further at the time of expected puberty. The diagnosis of 5 alpha-reductase deficiency was confirmed by demonstration of male levels of testosterone and testosterone precursors before and after hCG administration, elevated plasma testosterone to dihydrotestosterone and urinary etiocholanolone to androsterone ratios, and by in vitro studies indicating 5 alpha-reductase enzyme deficiency in the epididymis of one patient. Studies of control and mutant epididymal microsomes indicated that a single enzyme is responsible in the normal person for the 5 alpha-reduction of testosterone and cortisol (and probably other delta 4-3-ketosteroids as well) and that 5 alpha-reductase activity is undetectable for all substrates examined in the mutant. This finding explains why the formation of 5 alpha-reduced glucocorticoids is also defective in the disorder. | US |
We extracted human placentas by three methods to obtain chorionic TSH (hCT). The extracts were assayed by a sensitive homologous RIA for bovine TSH with an antibody which had veen used previously for assay of hCT. Based on the RIA for hCT, the yield of hCT was only 0.68 +/- 0.15 (SE) mU/100 g placenta by the Bates method, 2.01 +/- 0.35 mU/100 g by the Reisfeld method, and 4 mU/100 g by a Concanavalin A-Sepharose technique. Thyrotropic activity was detected by bioassay only in one Reisfeld extract; this biological activity could be attributed to hCG in this fraction. In TSH bioassays of other extracts, the responses were less than the sensitivity of the assay; some extracts were lethal to the bioassay mice. The low yield of hCT, about one-tenth to one-hundredth of that reported previously, casts doubt on the validity of the previous observations. | US |
A patient with hypopituitarism and an unusually large chromophobe adenoma which secreted an enormous amount of human PRL (hPRL) provided us with a unique opportunity to study the heterogeneity of hPRL. Serum samples obtained before and after surgery as well as pituitary extract were studied. Each sample contained three distinct hPRL peaks on gel filtration designated as big medium, and small. The hPRL moiety of each peak fraction was stable in size, immunoreactivity, and bioreactivity, indicating that the polymorphic hPRL components, once formed, were not interconvertible. As the relative proportion of medium and small hPRL components in all blood samples obtained before surgery and in the pituitary tumor extracts were similar, it seems that these two forms of hPRL originate from the tumor. However, there was a lack of correlation between big, medium, and small hPRL in samples obtained before and 1 yr after surgery. Also, increasing amounts of radioactive substance similar to increasing amounts of radioactive substance similar to big PRL were formed by exposure of 125I-labeled small PRL to progressively larger concentrations of serum. Thus, only small and possibly medium PRL are secretory products. | US |
The effect of flutamide on cortisol metabolism was studied in eight patients with prostate cancer. Flutamide markedly decreased the formation of 3 alpha, 17,21-trihydroxypregnane-11,20-dione (THF), and the 11-oxy-17-ketosteroid metabolites by 72%, 50%, and 46% respectively; however, 3 alpha, 11 beta, 17,21-tetrahydroxy-5 alpha- pregnan-20-one was increased by 46%. The 24-h mean plasma cortisol concentration was not altered. The cortisol production rate decreased by an average of 53% (from 32.7 to 15.5 mg/24 h). The effect of the drug on plasma cortisol kinetics was studied in three patients. This showed that flutamide increased the t1/2 (from 80 to 108 min) but decreased the distribution volume (from 17.8 to 13.8 liters) and the MCR (from 222 to 130 liters/24 h). The changes in THE and THF formation and in the t1/2 and MCR of [C]cortisol are similar to the effects observed in patients with intrahepatic cholestasis. It is suggested that in the case of flutamide these changes were also due to a cholestasis-producing effect of the drug on the liver. As the clinical response to the drug did not correlate with the cortisol metabolic changes, its therapeutic effect was probably not mediated by its effects on cortisol metabolism. | US |
The effect of an acute elevation of the serum magnesium concentration on the concentrations of serum immunoreactive parathyroid hormone (IPTH) were studied in hypocalcemic hypomagnesemic patients, hyperparathyroid patients, and normal individuals. Basal serum IPTH concentrations in the hypomagnesemic patients ranged from undetectable to 3 times the upper limit of normal. All hypomagnesemic patients were observed to have an immediate rise in the serum IPTH concentration after magnesium administration regardless of the basal IPTH concentration. In contrast, normal individuals and patients with primary and secondary hyperparathyroidism responded to magnesium administration with either a decrease or little change in the serum IPTH concentration. These date indicate that an acute stimulation of PTH secretion induced by magnesium is characteristic of the magnesium-deficient state. The consistency of this response suggests that impaired PTH secretion is a significant factor contributing to the hypocalcemia of magnesium deficiency. | US |
The metabolism of cortisol-21-glucosiduronate has been studied in two subjects. Urinary excretion was about 65% in both subjects, with cortisol glucosiduronate as the principal metabolite accompanied by small amounts of 11 beta, 17,21-trihydroxypregnane-3,20-dione (THF) glucosiduronate. The absence of the other normal metabolites of cortisol [17,21-dihydroxypregnane-3,11-20-trione (THE), 3 alpha, 11 beta, 17, 20xi, 21-pentahydroxypregnane, and 3 alpha, 17, 20xi, 21-tetrahydroxypregnan-11-one] indicates that negligible hydrolysis at C-21 occurred and that an unoccupied C21 position is required for oxidation at C-11 and reduction at C-20. The limited conversion to THF suggests that a pathway through cortisol-21-glucosiduronate does not contribute significantly to the differences in specific activity of urinary THF and THE observed after administration of labeled cortisol. | US |
[14C]Cortisol was injected iv into three subjects during a control period and while receiving metyrapone. The plasma kinetics of the tracer cortisol and the patterns of its urinary metabolites were measured. Metyrapone caused an increase in the volume of distribution of cortisol (34%) and in the MCR (75%); the half-life was decreased by 25%. There were marked changes in the urinary metabolite pattern: 3 alpha,11 beta,17,21-tetrahydroxy-5 alpha-pregnan-20-one, 3 alpha,17,21-trihydroxy-pregnane-11,20-dione(THE), pregnane-3 alpha,11 beta,17,20 alpha,21-pentol, plus pregnane-3 alpha,11 beta,17,20 beta-21-pentol (cortol), and 3 alpha,17,20 alpha,21-tetrahydroxypregnan-11-one (cortolone) all decreased by an average of 62%, 44%, 38%, 45%, and 25% respectively. In contrast, there was an increase of 296% in 3 alpha,17,20 beta,21-tetrahydroxypregnan-11-one (beta-cortolone). To account for these effects it is postulated that metyrapone has the following extraadrenal actions: 1) it inhibits the back reduction of cortisone to cortisol and 2) it stimulates the 20-ketosteroid reductase that converts THE to beta-cortolone. | US |
The effect of starvation on the peripheral metabolism of rT3 was evaluated in four obese euthyroid patients. During starvation, the serum rT3 concentration increased by 69% while the MCR of rT3 decreased in all four patients from control values of 96 +/- 23 (mean +/- SD) to 68 +/- 17 liters/70 kg . day, resulting in a slight increase in the mean production rate of rT3. These findings are in contrast to the marked decrease in T3 production rate associated with fasting, indicating that inner and outer ring deiodination of T4 can be varied independently. | US |
Seven hundred micrograms of T4 were injected into the amniotic cavity 24 h before delivery of five pregnant women scheduled for elective cesarean section at term. T4, T3, and rT3 concentrations were measured by RIA in amniotic fluid obtained at the time of the injection and in amniotic fluid and cord serum samples collected at delivery. Iodothyronine concentrations also were determined on cord samples from 24 full term control infants. The geometric mean serum T4 concentration in the experimental infants was 27.2 micrograms/dl, almost 3 times that of the control population (10.3 micrograms/dl); serum rT3 concentrations were markedly elevated to a mean of 657 ng/dl, compared to 254 ng/dl in control infants. The mean serum T3 concentration was slightly but significantly increased to 61.3 ng/dl (control, 48.3 ng/dl; P less than 0.02). Amniotic fluid T4, T3, and rT3 concentrations all increased significantly. T4 injection into the amniotic fluid is an effective method of increasing fetal serum T4 concentrations. The preferential pathway of monodeiodination of the injected T4 in the human fetus is to rT3 rather than T3. | US |
Specific RIAs requiring ether extraction only were established for estrone and 17 beta-estradiol both in plasma and in urine from the nonpregnant female. These assays were used to measure the renal clearance rates of estrone and of 17 beta-estradiol in eight ambulatory women in the follicular and in the luteal phases of the menstrual cycle. The mean (+/-SE) for the renal clearance rate of estrone was 0.71 +/- 0.058 ml/min in the follicular phase and 1.26 +/- 0.35 ml/min in the luteal phase. The mean (+/-SE) renal clearance rate of 17 beta-estradiol was 0.44 +/- 0.055 ml/min in the follicular phase and 0.29 +/- 0.043 ml/min in the luteal phase. There was no significant difference in the renal clearance rates of either estrone or of 17 beta-estradiol between the follicular and luteal phases of the cycle. The renal clearances of estrone and 17 beta-estradiol were highly correlated (r = 0.84; P less than 0.01). The renal clearance rate of estrone was significantly greater than that of 17 beta-estradiol in both phases of the cycle (P less than 0.01). | US |
Estrone sulfate, quantitatively the most important estrogen in plasma, has previously been determined only after hydrolysis and chromatography. An antiserum raised against estrone glucosiduronate-bovine thyroglobulin was found to be suitable for the specific RIA of estrone sulfate both in plasma and urine. Plasma levels were measured after solvent extraction without hydrolysis or chromatography. The mean (+/-SE) was 972 +/- 79 pg/ml (range, 537-1581) in 15 women in the follicular phase, 1806 +/- 160 pg/ml (range, 814-3358) in 15 women in the luteal phase, and 922 +/- 62 pg/ml (range, 461-1238) in 13 men. The urinary excretion of estrone sulfate, measured after simple chromatographic separation, ranged from 0.8-7.9 micrograms/24 h in men and 5.1-18.7 micrograms/24h in nonpregnant women. This was generally one-seventh to one-half the simultaneous estrone glucosiduronate excretion rate. An approximate mean renal clearance of estrone sulfate calculated from the above values was 2.7 ml/min. The low clearance rate is taken to reflect extensive binding of estrone sulfate by plasma proteins. The splanchnic extraction of estrone sulfate measured in 6 patients undergoing hepatic vein catheterization for diagnostic purposes was 29.8 +/- 11.1%, indicating net uptake of this compound by the splanchnic area. | US |
The regulation of both the activity of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase [mevalonate-NADP+ oxidoreductase (CoA-acylating) EC 1.1.1.34] and the secretion of progesterone by human plasma lipoproteins has been investigated in human choriocarcinoma cells in culture. HMG CoA reductase activity was computed from the rate of formation of [14C]mevalonolactone from [14C]HMG CoA. The activity of HMG CoA reductase was expressed as nanomoles of mevalonolactone formed/min . mg solubilized cell protein. An inverse relationship was found between the presence of lipoprotein in the culture medium and the activity of HMG CoA reductase in these cells. In cells maintained in the presence of lipoprotein-enriched culture medium containing 840 micrograms cholesterol/ml, the average activity of HMG CoA reductase was 0.25 nmol/min . mg protein. After removal of lipoprotein, the activity of HMG CoA reductase increased to 1.3 nmol/min . mg protein. The average activity of HMG CoA reductase in cells maintained in lipoprotein-deficient culture medium was 1.5 nmol/min . mg protein but fell to 0.3 nmol/min . mg protein after addition of lipoprotein to the medium. When cells were maintained in the presence of lipoprotein, the rates of section of progesterone and pregnenolone into the culture medium were 2-8 times greater than the rates of secretion of these steroids by cells maintained in the absence of lipoprotein. On the basis of these results, it is concluded that lipoproteins control the rate of cholesterol biosynthesis in cultured choriocarcinoma cells by regulating the activity of HMG CoA reductase, and control the rate of synthesis of progesterone by providing the precursor, cholesterol. We suggest that progesterone synthesis by the trophoblast of the human placenta may also be regulated by the uptake of lipoprotein from maternal blood. | US |