Stimulation of albumin synthesis by keto analogues of amino acids. (1) The effect of ketoanalogues of branched-chain amino acids on albumin synthesis was examined in two biological systems using the [14C]carbonate technique. (2)alpha-Ketocaproic acid, the ketoanalogue of leucine, was able to reverse the reduced synthesis rate observed when isolated livers, from well-nourished animals were perfused with blood from rats deprived of dietary protein for 48 h. (3) A mixture of ketoanalogues of the three branched-chain amino acids, leucine, isoleucine and valine, was able to increase albumin synthesis per unit dry liver weight to above normal levels when administered intragastrically to rats 16 h after partial hepatectomy.


The formation and annealing of structural defects in lipid bilayer vesicles. It is shown that sonication of phospholipid-water dispersions below the crystalline leads to liquid crystalline phase transition temperature (Tc) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above Tc. The rate of annealing was found to be slow, of the order of an hour for T = 3 degrees C above Tc, but annealing is complete within 10 min for T = 10 degrees C above Tc. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside in equilibrium outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 degrees C above the phase transition.


Standing-gradient osmotic flow. Examination of its validity using an analytical method. The solutions to the non-linear differential equations governing solute-solvent coupling in the intercellular spaces of epithelial layers have been obtained by using an analytical method, rather than the usual numerical ones. When the present series solution includes second-order correction terms, the concentration and velocity profiles obtained by the analytical method agree very well with those coming from numerical solutions. This method has further allowed us to examine the standing-gradient hypothesis when applied to the backwards fluid transport system of the corneal endothelium. With the information presently available for the relevant parameters (osmotic permeability, rate of transport, radius and length of the spaces, and location of the pumping sites), near-isotonicity of the transported fluid would not be explained by the standing-gradient model.


Biochemical studies on abnormal erythrocyte membranes. Protein abnormality of erythrocyte membrane in biliary obstruction. Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken, By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.


Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes. Phenol extraction of horse, sheep, cow, pig and human erythrocyte membranes and human milk fat globule membranes gave glycoprotein fractions, all of which were shown by gas chromatography to contain the reduced disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosaminital after treatment with alkaline borohydride. Cow and pig erythrocyte membrane glycoproteins were found however to contain much lower amounts than the erythrocyte membrane glycoproteins of the other species tested. After gel filtration, a tetrasaccharide was isolated from horse and sheep glycoproteins containing the disaccharide plus two molecules of sialic acid. Periodate oxidation together with paper chromatography of alkaline degraded fragments showed these two molecules of sialic acid to be linked to positions C3 and C6 of the galactosyl and N-acetylgalactosamine residues respectively. Evidence was obtained for a similar structure from pig and cow erythrocyte glycoproteins and human milk fat globule membrane glycoproteins although the complete structure was not elucidated. In all native glycoprotein fractions, the unsubstituted disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosamine was found to be present to different extents. Haemagglutination inhibition tests against human anti-T serum, Arachis hypogoea and Vicia graminea by desialylated glycoproteins showed the presence of the T-antigen, confirming the chemical findings. Inhibition was found to be proportional to the chemically detected amounts of disaccharide in each fraction. Evidence for a second carbohydrate chain in horse, sheep and human erythrocyte glycoproteins with a sialic acid substituted N-acetylgalactosamine residue as the terminal sequence was obtained using the agglutinin from Helix pomatia.


Preparation and characterization of calcium-binding and other hydrophobic proteins from synaptic membranes. A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.6 - 10(4), a binding capacity of 4 calcium/molecule, and a Km of 1.5 - 10(-5) M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides.


The chromaffin granule surface. Localization of carbohydrate on the cytoplasmic surface of an intracellular organelle. Intact chromaffin granules from bovine adrenal medulla are shown to have complex carbohydrates on their external (cytoplasmic) surface. This is demonstrated by the facts (1) that granules can be agglutinated by wheat germ agglutinin, and (2) that significant amounts of sialic acid can be removed from the granule surface with neuraminidase. Glycoproteins located in the granule membrane, and not glycolipids, are the molecules that mediate wheat germ agglutinin agglutination. The possible involvement of granule surface carbohydrate in the process of exocytosis is discussed.


The affinity of rat liver ribosome subunits for degranulated rough microsomal membranes. Potassium and magnesium ion concentrations affected the extent but not the specificity of binding in vitro of 60-S and 40-S ribosome subunits to degranulated rough microsomal membranes from rat liver. Scatchard plots revealed that under ionic conditions most likely to resemble those in vivo, the affinity constants for binding 60-S subunits were approximately four-times greater than those characterizing 40-S subunit binding. Further, the extent to which subunits bound at saturation was close to the level of ribosomes present in intact membranes.


Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia. Membrane potential, Vm, and K+ (86Rb+) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. -170 mV) the K+ influx amounts to 0.24-0.6 pmol-cm-2-s-1 and the K+ efflux to 0.2-1.5 pmol-cm-2s-1. According to the K+ concentrations inside and outside the cell (40:1) the voltage dependent K+ flux (zero at Vm = EK = -90 mV) is stimulated approx. 40-fold for Vm more positive than EK. It is calculated that during one action potential (temporary depolarization to Vm more positive than EK) a cell looses the same amount of K+, which leaks in during 10-20 min in the resting state (Vm = -170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K+ balance of this alga.


Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by a 4-azido-2-nitrobenzoate derivative of phlorizin. Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO42- equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO42- exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular tritiated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccessibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.


Effects of electrical gradients on volume flows across gall bladder epithelium. A volumetric method has been developed which permits continuous registration of volume flows across epithelial tissues. The method was applied to volume flow measurements across rabbit gall bladder epithelium. The rate of fluid reabsorption measured in this way was twice as high as previously observed in sac preparations of the gall bladder. This is probably due to better aeration and stirring of the mucosal solution. It was demonstrated that electrical gradients across the gall bladder induced volume flows towards the negative electrode. In non-transporting bladders volume flows were linearly related with current between 300 and 900 muA in both directions. However, volume flow rates were three times higher from mucosa to serosa than in the opposite direction. From the magnitude of polarization potentials, observed after switching off the current, the conclusion was reached that all of the current-induced volume flow is an osmotic flow due to salt polarization in the unstirred layers of the tissue. By implication, so-called streaming potentials observed during osmotic flows reflect solely polarization effects. In actively transporting gall bladders a 200 muA current increased or decreased the flow rate twice as much as expected from polarization effects alone. Therefore passage of current interfered directly with the active transport mechanism of gall bladder epithelium.


Oxidative damage of retinal rod outer segment membranes and the role of vitamin E. Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.


Interaction of concanavalin A with rabbit thymocyte plasma membranes. Distinction between low affinity assoication and positively cooperative binding mediated by a specific glycoprotein. 1. We have analyzed the interaction of the mitogenic lectin, concanavalin A, with purified plasma membranes isolated from rabbit thymocytes. 2. Scatchard analyses show that in native membranes binding is positively cooperative at low concanavalin A concentrations and non-interacting at high lectin levels. 3. In contrast, membranes treated with 0.0064 M glutaraldehyde exhibit diphasic Scatchard plots, indicating the presence of high- and low-affinity binding sites. The high-affinity zone corresponds to the region of positive cooperativity in native membranes. 4. The number of high-affinity binding sites per cell-equivalent corresponds approximately to the number of glycoprotein (mol. wt. 55000) molecules (1-10(6)/cell), but account for less than 25% of the total lectin binding. 5. Treatment of membranes with 0.0064 M glutaraldehyde selectively crosslinks the glycoprotein (mol. wt. 55000) and its multimers, correlating directly with the modifications of concanavalin A-binding. 6. We conclude that high-affinity binding of concanavalin A to thymocyte membranes is a cooperative process mediated by the glycoprotein (mol. wt. 55000). We further conclude that the bulk of concanavalin A binding is through low-affinity associations, not involving specific membrane macromolecules.


Deuterated fatty acids as Raman spectroscopic probes of membrane structure. Raman spectra are reported for the C-D stretching region of stearic acid-d35 bound in egg lecithin multilayers. The temperature dependence of the spectra shows that the linewidth of the C-D stretching bands is a sensitive and non-perturbative probe of membrane hydrocarbon chain conformation. The utility of this approach for studying lipid conformation in membranes containing a significant fraction of non-lipid component is discussed.


A measurement of the proton pump current generated by bacteriorhodopsin in black lipid membranes. The light-induced electrical current generated by black lipid membranes containing bacteriorhodopsin from Halobacterium halobium has been measured directly. It is shown that a measurement of membrane potential can also be used to obtain the proton pump current developed during illumination. Evidence is presented that the charge movement across the membrane is associated with the release of protons in the photoreaction cycle of bacteriorhodopsin. The time variation of the pump current when the light is turned on suggests the rapid depopulation of some initially occupied state.


Synthesis and turnover of leghaemoglobin in lupin root nodules. 1. The problem of whether leghaemoglobin is synthesized on plant of bacterial ribosomes in root nodules of yellow lupin has been examined. 2. Leghaemoglobin, soluble plant protein and soluble bacteroid protein were labelled with 14C administered by uptake of 14CO2. 3. Exposure of roots to 1 mM D-threo-chloramphenicol resulted in inhibition of soluble bacteroid protein synthesis, but leghaemoglobin synthesis and soluble plant protein synthesis were unaffected. This result is consistent with leghaemoglobin being synthesized on plant ribosomes. 4. After nitrogen-fixing plants had been supplied with a pulse of 14CO2, the decay of specific radioactivity of nodule protein fractions was observed. Leghaemoglobin had an apparent half-life of 18 days and is a stable protein in nitrogen-fixing yellow lupin nodules.


Molecular weight determinations on the alpha-globin and beta-globin mRNAs. The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A)  regions which occur as the mRNAs mature.


Preferential regulation of protein synthesis initiation complex formation by purine nucleotides. A comparison of the affinities of eukaryotic initiation factor 2 and eukaryotic elongation factor 1 for GTP and GDP, and of the responses of initiation and elongation complex formation to various GTP mol fractions indicated that the initiation reaction was more sensitive to changes in the GTP: GDP ratio. In vitro regulation of the GTP: GDP ratio by the adenylate energy charge, a sensitive control parameter, also demonstrated a preference for regulation of formation of initiation complexes when compared to elongation complexes. These studies suggest that, based on the availability of energy, initiation is the rate-limiting step in the overall protein synthetic process.


Protein synthesis in chloroplasts. VII. Initiation of protein synthesis in isolated intact pea chloroplasts. Isolated intact pea chloroplasts use light energy to synthesise N-formayl [35S]-methionylpuromycin when incubated with L-[35S]methionine and puromycin. Control experiments establish that this synthesis occurs on chloroplast ribosomes, and not on contaminating mitochondrial or bacterial ribosomes. The amount of N-formylmethionylpuromycin formed suggests that each messenger RNA that is being translated in vitro undergoes initiation at least twice. We conclude that isolated, intact pea chloroplasts carry out the initiation of protein synthesis, as well as the elongation and termination of polypeptide chains.


Ribosomal RNA synthesis in the mycelium of Phycomyces blakesleeanus. High molecular weight RNA. Total RNA was extracted from 24 h old mycelia of Phycomyces blakesleeanus which had been grown in liquid culture. Heavy molecular weight RNA was fractionated on 2.6% polyacrylamide gels. 1. The main cytoplasmic ribosomal RNA fractions had molecular weights of 1.34 and 0.72-16(6). 2. [3H]Uridine pulse labelling of cultures revealed an initial ribosomal RNA precursor of molecular weight 2.5-10(6). 3. An intermediate precursor for the 1.34 - 10(6) dalton peak was found, having a molecular weight of 1.6-10(6). 4. Pulse-chase experiments, using [3H]methionine, showed the flow of synthesis as being (see article). 5. Mature 0.72-10(6) dalton RNA appeared prior to the 1.34-10(6) dalton RNA but no immediate precursor for it was detected.


Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells. We have previously presented evidence that hydroxyurea treatment of synchronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Walters, R.A., Tobey, R.A. and Hildebrand, C.E. (1976) Biochem. Biophys. Res. Com. 69, 212-217). In view of the reported effects of hydroxyurea on deoxyribonucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248,3904-3909 and Walters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336-347), we examined the fate of DNA synthesized during and shortly after hydroxyurea treatment to determine if this DNA exhibited any kinetic behavior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 +/- 0.12 - 10(6) dalton/min (0.98 +/- 0.12 mum/min) for about 2.3h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 - 10(8) was very rapidly integrated into bulk DNA of greater than or equal to 3.5 - 10(8) daltons. The apparent growth rate of the 1.4 - 10(8) dalton DNA was approx. 10.6 mum/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and once begun, occurred very rapidly. The possibility of inegration of replicon clusters is considered.


Histone inhibition of DNA synthesis in eukaryotic cells permeable to macromolecules. Mouse L Cells, grown in suspension culture can be rendered permeable to exogenous deoxynucleoside triphosphates by a cold shock in a near isotonic buffer system. These cells use the deoxynucleotides to synthesize DNA in a semiconservative fashion. The addition of 0.05% Triton X-100 to this system increases the permeability of the cells so that exogenously supplied macromolecules gain access to the DNA. When DNAase and phosphodiesterase are added to the detergent-permeabilized cells, the cell DNA is rapidly degraded, demonstrating that the enzymes reach the DNA within the first 2 min of the incubation period. Addition of whole calf thymus histone or histone fractions to the detergent-permeabilized cells inhibits DNA synthesis. The lysine-rich histone, F is a more effective inhibitor than the arginine-rich histone, F3. The other histone fractions including the slightly lysine-rich fractions, F2a and F2b, are intermediate between F1 and F3 as inhibitors of DNA- synthesis. Kinetic analysis demonstrates that the added histones increase apparent Km and reduce V of DNA synthesis in the permeabilized cells. These studies suggest the possibility that histones alter the association of the DNA replication complex and the DNA template in a manner that reduces the rate of DNA synthesis.


Double strandedness of nascent DNA of a higher plant (Vicia faba). After a pulse of 5-10 min with [3H]thymidine, labeled DNA extracted from embryonic axes of Vicia seeds sedimented as a rather homogeneous peak at approx. 10S in an alkaline sucrose density gradient, as described in our previous paper ((1975) Biochim. Biophys. Acta 395, 314-321). The sedimentation pattern of the same pulse-labeled DNA in a neutral gradient showed a wide range of sizes from approx. 14 S to more than 40 S. However, most of these labeled DNA components, including the 14-20 S shorter fragments, were shown to have a double-stranded structure by hydroxyapatite column chromatography. Further investigations on neutral sucrose gradients revealed a decreased occurrence after a chase, and final disappearance after a longer chase period of these shorter double-stranded fragments. A possible secondary structure of the newly synthesized DNA in a higher plant (Vicia faba) is discussed.


Free and membrane-bound ribosomes. VI. Distribution of free sulphydryl groups in rabbit reticulocyte ribosomes. When free and membrane-bound ribosomes were titrated with N-ethyl[14C]-maleimide or 5,5'-dithio-bis(2-nitrobenzoic acid), 25 free SH groups were detected in  polysomes, 10 in the 60 S subunit and 14 in the 40 S subunits. When individual ribosomal proteins were analysed, one large subunit protein L14 was found to be strongly labelled compared to the other ribosomal proteins, in polysomes, monosomes and subunits. However, two additional protein spots from the small subunit were more radioactive in monosomes and subunits than inpolysomes. In all these studies, it was found that the distribution of free SH groups in free and membrane-bound ribosomes was identical except for the few proteins which were reported before to be specific for free or membrane-bound ribosomes (Fehlmann, ., Bellemare, G. and Godin, C. (1975) Biochim. Biophys. Acta 378, 119-124 and Fehlmann, M., Bellemare, G. and Godin, C. (1975) FEBS Lett. 59, 8-12).


[Distribution of identical amino acid residues in the primary structure of proteins]. It is shown that the distribution of identical amino acid residues in the primary structure of 83 non-homologous proteins containing about 14 000 amino acids is near to a casual one, i.e. it is determined by the amino acid composition. A characteristic feature of such distribution is an increased probability of a common grouping of amino acid residues.


[Selective broadening of NMR lines as a criterion in studying the interaction of Mn(II) with amino acids and peptides in D20]. A proton NMR study was carried out on complexes of Mn(II) with amino acids and peptides in aqueous solution. The theory, based on the Solomon-Bloembergen equations, was summarized and discussed. Different types of equilibria involving the anionic and zwittrionic forms of the amino acids were taken into account, together with the presence of outer- and inner-sphere species. The related correlation times were evaluated. Sarcosine, beta-alanine, threonine, tyrosine, valine, beta-alanine, proline, histidine and cystein complexes with Mn(II) were investigated on the basis of the NMR spectra. Moreover, diglycine and triglycine were studied in the presence of manganous ion at different pH value. The findings were discussed in terms of selective broadening of the NMR peaks and compared with data in the literature.


[Optical measurements of nucleic acids in the presence of phenol and protein impurities]. Spectrophotometric method of determining the presence of phenol admixture in nucleic acid samples is presented. The method is based on determining the optical density ratio at 270 and 240 nm. This ratio is highly sensitive to the presence of phenol in solution. The method of determining nucleic acid concentration in the presence of phenol is also described. It is based on recording circular dichroism values at 265 nm for RNA and 275 nm for DNA. It is shown that the ratio of optical densities of nucleic acid samples at 260 and 280 nm is not a very sensitive criterion of the presence of proteins in solution. A much more sensitive characteristics is the ratio of circular dichroism values at 222 and 265 nm.


[Formalin-induced chemiluminescence in model protein-lipid systems]. Mechanisms of formalin--induced luminescence of protein-lipid complexes have been studied. Free radical reactions of lipid oxidation were shown to be necessary for generating luminescence quanta. The luminescence intensity was mostly promoted by fluorescing products formed in the reoxidized system protein-lipid. Formalin-induced flash of luminescence in these systems may evidently serve as an express-method for analysing protein sample contamination with unsaturated lipids.


[Determination of the mechanism of action of antioxidants in lipid systems from chemiluminescence in the presence of ferrous oxide]. Different action mechanisms of antioxidants affecting various stages of chemoluminescence developing in the lipid systems with ferrous ions are considered theoretically. Experimental studies are carried out on beta-naphtol and 5-oxitriptamine--antioxidants whose action mechanism in other systems has been investigated. It is concluded while comparing the experimental data with theoretical concepts that beta-naphtol in mitochondria and liposome suspensions binds the radicals according to the "sticking" mechanism, while 5-oxitriptamine reduces hydroperoxides.


[The simplest biochemical autogenerator--open enzyme reaction, S in equilibrium with P, with substrate inhibition]. Considered is the mathematical model of an open reversible enzymic reaction S equilibrium P with enzyme E inhibited by excess substrate S. The quasi-stationary output characteristic of the reaction, v(p), is shown to be of hysteretic Z-shaped form. It gives the rate of the product formation, v, as a function of of p=P], provided d[S]dt=0. Owing to hysteresis of the characteristic the reaction can exhibit 1 to 3 stationary states of different stability. Under specified conditions there occur self-oscillations in the reaction. Multiple stationary states and self-oscillations take place only with positive v, i.e. when the net flux is from S to P. The reaction considered is the simplest of all biochemical oscillators studied thus far.


[Oxidation-reduction potentials of chlorophyll pigments in photosynthesizing organisms on different levels of evolutionary development]. According to polarographically measured EOred of bacteriochlorophyll "a", chlorobium chlorophyll "660", "b" and "a" chlorophylls (-0.67 V, -0.73 V, -0.79 V, -0.86 V-n.h.e.) and literature data about Eoox of these pigments (+/-0.62 V, + 0.62 V, + 0.78 V, +0.77 V correspondingly) it has been shown that chemical energy stored in the singular photochemical set is increased in the evolution of the photosynthetic apparatus. It has apparently resulted in the possibility of evolutionary approach to utilization of more inert and more accessible (water) electron donors and favoured further development of photosynthesizing organisms.


[Experimental verification of a mathematical model of plasma protein and erythrocyte regeneration after blood loss]. The regeneration process after 40% bleeding is mathematically regular. Albumin, hemoglobin and red cells regeneration is expressed by the fraction-linear function. This law is true for albumin resotration only immediately after blood loss. Red cells and hemoglobin recovery begins only 3 days after acute bleeding, when albumin concentration rises by 90% of the normal level. Mathematical regularity of albumin regeneration was destroyed after the liver denervation or spleenectomy.


[Biopolymer crystallization in malignant tumors]. It is shown by statistical analysis of structural studies of different polymers and polypeptides compounds, qualitative considerations and experimental studies of ascite strain of Ehrlich tumour that the growth of the tumour is accompanied by a specific course of crystallization of biopolymers.


[Structural changes in ganglioside micelles during interaction with serotonin]. Structure changes of ganglioside micelles in the process of serotonin bonding are considered. It was shown by viscosimetric and differential refraction studies that stretching of elliptical ganglioside micelles in the serotonin bonding process took place. The scope of micelles form changes depends on the pH value. An increase of the micelles summary negative charge at pH 9 increased the number of bonding centres and simultaneously caused deeper deformation of micelles due to the repulsion of the identically charged groups.


[Distribution of small hydrophobic molecules in membranes. II. Heterogeneity of binding centers on a phospholipid bilayer surface]. 4-dimethylaminochalcone (DMC), a hydrophobic uncharged fluorescent probe, was bound to a phospholipid bilayer surface and was distributed in different binding sites. Sites whose polar groups of phospholipid and hydration shell molecules were the least mobile had the greatest affinity to DMC. The increase DMC/phospholipid ratio resulted in DMC molecules getting bound to sites of a lesser affinity whose polar groups were more mobile. Cholesterol presence caused DMC binding to the first type of sites only.


[2 mechanisms of membrane electroconductivity with tetrachloro-2-trifluoromethylbenzimidazole (TTFB)]. It has been found during galvanostatic measurements under the conditions of diffusion overtension that charge transfer through the membrane is carried out by the transfer of a proton from a neutral form to an anion (acidic region), or by transfer of a negative charge--dimer due to the formation of a hydrogen bond between them on the membrane border (alkaline region).


[Potential difference across subcellular particle membranes. I. Chemiosmotic and chemielectric mechanism of generation]. Transformation of light energy, of substrate oxidation and ATP hydrolysis energy into electric form during both oxidative and photo-phosphorylation can be described not only by means of chemiosmotic but also by chemielectric hypothesis. The latter hypothesis supposes, that dehydrated protons are taken up by electrostatic forces into the inner part of the membrane. Protons move into the input channels following electrons driven by chemical forces. Inside the membrane H+-ions are released into the output channels when electrons are transferred to the next electron carriers. Output H+-channels eject protons towards the other side of the membrane along the gradient of rising polarization of the channels. The inner resistance of chemielectric potential generators is lower than that of chemiosmotic one. A model of chemielectric mechanism is proposed. According to this mechanism electrons driving protons move along nonheme iron proteins and H+-ions are released into the output channels from semiquinones and hydroquinones.


[Potential difference across subcellular particle membranes. II. Compensation method of measurement]. "Zero-loop" of the molecular potential transformer of submitochondrial particles (SMP) is separated from the remaining electron transfer chain by rotenone, and its e.m.f. ET=0,003+RT/2F in [NADP X H] [NAD+]/[NADP+] [NAD X H] volts is used in the compensative method of measurement of the potential difference across the SMP membrane (delta USMP). The phospholipid membrane, measuring the concentration of the penetrating anions in the solution contained SMP, is used as "zero-indicators". This concentration drops monotonically with increase in delta USMP. Delta USMP is equal to ET when the addition of substrates of transhydrogenase reaction with definite ET does not change the potential across phospholipid membrane.


[Spectroscopic study of the interaction of cationic dyes with mitochondria]. Data are presented on the changes of absorption spectra of two cation dyes: nils blue and acridine orange while binding them with intact mitochondria of the rat liver in various functional states. It is found that when the dyes are bound with mitochondria characteristic changes proceed in the absorption spectra of dyes. These changes show that the formation of the dye molecule dimers is more intensive on the membrane than in solution. Energisation of mitochondria leads to a more intensive formation of acridine orange dimers, but the formation of nils blue dimers becomes less intensive. A suggestion is made about different nature of the interaction between these dyes and active groups of the mitochondria membrane.


[Eserine-induced change in membrane potential of frog muscle fibers]. The action of eserine of acetylcholinesterase (AchE) reversible inhibitor of frog muscle fibres membrane potential (MP) under various physico-chemical conditions in external solution was studied. The data obtained show that changes of external pH in any direction decrease the depolarisation of the membrane produced by eserine. The dose-effect curve is linear at pH 7, but it has saturation at pH 6 and pH 9. Dependence of the membrane depolarisation in the presence of eserine upon calcium ions concentration in external solution is S-shape. Protonophore 3C1CCP (carbonilcianamid-m-3C1-phenylhydrazon) depolarises the membrane further in the presence of eserine. Valinomycin under these conditions completely restores the MP. Evidence is obtained that eserine reduces potassium permeability of the muscle membrane. It is supposed that membrane-bound AchE is involved in the ionic permeability regulation of the muscle membrane at rest.


[Are the reciprocal sliding of protofibrils and the shortening of thick filaments during muscle contraction based on a common molecular mechanism?]. It is assumed that the back stroke of myosin bridges in a contracting fibre is determined by nucleotide binding. Then an inhibition of actomyosin dissociation can lead to the shortening of the thick filaments. Possible existence of a protein control system, suppressing this dissociation is discussed.


[Inactivation of skeletal muscle actin from newborn and adult rabbits in different conditions of polymerization and sample storage]. Changes in polymerization degree of actin of newborn and adult rabbit muscles while storing solutions were studied. It is found that under different polymerization and storage conditions including the presence of ATP, Ca++ ions, 2-metacaptoethanole of ethanole which slow down the inactivation of actin the latter proceeds faster in newborn rabbits than in adult ones. It is suggested that with different inactivation rate of actin of newborn and adult rabbits are associated the differences in their polymerization degree found earlier.


[Deformation and morphogenesis of Acetabularia cell membrane. I. Relation of deformation to genetic and geometric factors]. Relationships between the changes of geometric shape, volume and deformations of the apical growth part of acetabularia cell are studied. Jung module E approximately 25 kg/cm2, viscosity coefficient of the apex cell theta 4 - 10(4) kg-sec/cm2 and intracellular pressure P approximately 1 kg/cm2 are measured. Cell shape formation according to Waddington is concerned with simple physical factors which are under the genetic control. It is shown that uneven deformations with the loss of shape stability bring about periodic cell complications.


[Relation of light-induced fluorescence quenching of Atabrine to photosynthetic electron transport in isolated pea chloroplasts]. Light-induced fluorescence quenching of atebrin in isolated pea chloroplasts was studied. The observed quenching effect was shown to be influenced by DCMU, ADP+P and cofactors of photosynthetic electron transport. In the presence of ADP+P the amplitude of the quenching in control plants was not changed, whereas in the pea mutant blocked at PS2 it was considerably decreased. However the dark decay rate of the quenching effect in both cases increased 2-3 folds.


[Deformation and morphogenesis of Acetabularia cell membrane. II. Local mechanisms of regulating viscoelastic deformations]. Relationships between growth velocities of some parts of Acetabularia cell, ratio between the thickness of the cell membrane and apex diameter (its constructive rigidity), intracellular pressure and viscose-elastic deformations are used for physical substantiation of Waddington's concepts concerned with the existence and important search of local morphogenetic mechanisms. Their realization in a strongly deformated regions of Acetabularia cell membrane is shown. The relationship between viscosity, synthesis of the material and dimensions of region and intracellular pressure is found.


[Asymmetrical behavior of small intestine epithelium under osmotic pressure]. The influence of wide interval of osmotic gradients on transepithelial potential differences through small intestine were investigated. It was found, that ion permeability begins to rise at osmotic pressures equal to 0.200-0.250 osmomol on serosal side and 0.350-0.400 osomomol on mucosal side. The variatin in behaviour of osmotic potential differences caused by flows directed from mucosal solution to serosal one and in the reverse direction at smaller osmotic pressure is shown. An assumption was made that observed features of behaviour of osmotic potential differences were due to structural reorganization in intracellular junction.


[Electric parameters and equivalent schematics for Acetabularia cells]. Was measured values of electric resistance (R) and capacity (C) of A. mediterranea cell at different levels of the membrane potential (MP). At the level -170 mV, RRP=4,3 KOm-cm2, at the level -80 mV REP=1,4 KOm-cm2. At both levels of MP C congruent to 1,3-10(3) mkF/cm2. A variant of equivalent electric scheme of the cell is suggested. Values of the elements comprising the scheme are presented, which allow an approximate reproduction of the value of MP levels and of transient processes between them.


[Relation between light-induced intracellular and extracellular bioelectric reactions of plants]. The parallel registration of light-induced changes of electrical potential of the water plant Elodea has been made with the help of intracellular-microelectrode and extracellular-contact methods. It has been shown that the curve of the light-induced bioelectrical reaction (LBR) under the extracellular registration was similar to the curve of the first differential of light-induced changes of intracellular electrical potential (LIP), but with a negative sign. Thus, the kind of changing of LBR, as the first differential of LIP, may give information about intracellular potential of ground plants, for which the microelectrode way of registration was impossible. The likeness between LBR of Elodea and the ground plants studied suggests the general regularities in the light-induced changes of electrical potentials of different plants.


[Work of large information systems].

[Work of large information systems]. The problem of application of informational ideas to the study of large informational systems, such as brain, is considered. Analysis follows some ideas of Winograd and Kowan's theory of reliable computations in the presence of noise. In the beginning of paper the "material analogy" for intuitive understanding of Shannon's results is proposed. Afterwards there is given the formulation of coding theorem for the case of "informational collective"--a large group of informational sources and receivers. In conclusion it is shown that such properties of neuronal structures as "stochastic" neural connections are reasonable from the point of information theory.


[Boundary conditions for excitation propagation equations which do not distort the picture of impulse movement]. New boundary conditions for the equations of excitation propagation are suggested. These conditions prescribe in the point in the end of the fiber the action potential which would be developed there, if half-infinite fiber would proceed further. The conditions suggested 1) in a number of cases allow to obtain an undistorted picture of excitation propagation, e.g. the picture when impulses fall out when a chain of impulses moves along the fiber; 2) save computer time and memory. Boundary conditions for syncitium and flat medium are considered.


[Modelling the reaction of the horizontal cell layer to non-uniform light]. A mathematical model of horizontal cells in fish retina is constructed. Some conclusions and hypotheses concerning potential distribution and synaptic current are discussed.


[Calculation of wave velocity in an excitable medium]. Analytical descriptions for the form and velocity values of sharp fronts in the impulse propagating in excitable medium are derived. In initial equations the form of nonlinear function is prescribed by the polynom of the third of fifth power.


[Torsional movements of the human eye. I. Torsional movements on inclining the head towards the shoulder]. The characteristics of the torsional eye movements during head tilting were investigated. Different conditions were used in the experiments. The subjects were in sitting and lying positions in the light and in the dark. A new method of recording with the suction device of Yarbus and the simple optical system allowed to have records without some redundant components of movement. This made record processing less complex and more accurate. The torsional drift in the slow phase of the eye movement was found present under any conditions. When being recorded simultaneously both eyes in man were in identical and synchronous movement.


[EEG potential theory in a model with thin brain integuments. III. Source--tangential double layer in the cortex]. Estimates of EEG-potentials obtained in the model of polylayer spherical and flat cables indicate that the geometry of the skull has only little influence. Potentials of EEG of the radial dipole and the tangential double layer are expressed by simple formules in these models. A theoretical explanation concerning great dispersion of ECoG to EEG ratio and identity of EEG-potentials, registered by two electrodes spaced within about 2 cm of one another, is presented.


[Moments in human leg joints during walking]. An automated method is developed to obtain the moments of internal forces in the leg joints during walking. A considerable variability of joints moments is observed, which sufficiently exceeds that of the kinematic parameters of walking. For normal walking three different types of joints moments can be distinguished. The formal foundation for the variability of the moments is given, and a hypothesis is proposed, according to which the variability is associated with the keeping of equilibrium.


CSF amine metabolites, clinical symptoms, and body movement in psychiatric patients. The relationships between CSF monoamine metabolites (HVA and 5HIAA), nurses' ratings of clinical symptoms, and telemetered measures of motor movement of ten schizophrenic and ten depressed patients were investigated. There was a significant negative correlation between CSF 5HIAA and both agitation ratings and motor movement in the schizophrenics. CSF HVA correlated positively to anxiety and anger in the depressives. The schizophrenics had a significantly higher CSF HVA than the depressives which appeared unrelated to motor movement. The effects of serotonin turnover and arousal in schizophrenia and the association between CSF metabolite gradients, stress, motor movement, and biogenic amine levels in depression are discussed.


Alcoholism: averaged visual evoked response amplitude-intensity slope and symmetry in withdrawal. Alcohol withdrawal has been characterized in terms of some EEG changes but seldom in terms of the averaged evoked response. Characteristics of the AER, such as amplitude, latency, and the least-squares slope of the AER amplitude as a function of stimulus intensity, have been found to change as a function of arousal and to separate some psychiatric groups. Seventeen alcoholics in withdrawal, 27 stabilized alcoholics, and 30 control volunteer subjects participated in an experiment to determine the changes in AER characteristics in withdrawal. Four stimulus intensities were presented to the subjects from a Grass PS 22 photostimulator with the flash sources 102 cm from the nasion. AER amplitude, the slope of the amplitude as a function of stimulus intensity, and bilateral symmetry scores were calculated for group comparison. Withdrawal and stabilized alcoholics were found to have significantly greater AER amplitudes at all intensity levels. Greater A-I slope from the right hemisphere and higher symmetry were found for withdrawal alcoholics than the other two groups.


Monoamine oxidase inhibitors: potential for drug abuse. Both the amphetamines and MAO inhibitors share common clinical and pharmacological properties, namely, (i) to clinically induce euphoriant-stimulating type and psychotomimetic effects in certain individuals, and (ii) to increase, albeit by different mechanisms, the amount of functionally available neurotransmitter (catecholamines and indoleamines) at the receptor site. The present data now indicate that, like the amphetamines, the use of MAO inhibitors can be clinically associated with dependence-tolerance. Perhaps these clinical findings will converge with other clinical-biochemical data in helping to define the specific amine(s) responsible for not only the clinical effects of these drugs but also the etiopathogenesis of major psychiatric illnesses such as the affective disorders and schizophrenia.


Subjective wellbeing and temporal patterns of sympathetic-adrenal medullary activity. Sixteen male subjects performed an audiovisual-conflict during 1 h of an ordinary day of work. Adrenaline and noradrenaline excretion and subjective reactions were measured before, during, and after the test period as well as at corresponding points in time in a control day of ordinary work with interruption. This program was carried out on two occasions, which were expected to differ with respect to the subjects' general mental condition and wellbeing. There were large intraindividual as well as interindividual variations in the rate at which adrenaline output decreased after the stress period. The results support the hypothesis that rapid recovery to baseline levels is positively related to a relaxed, alert and subjectively pleasant state of mind.


Organismic, stimulus and task determinants of phasic and tonic heart rate and skin conductance changes. The effects of three variables (perceptual style 'leveling--sharpening', stimulus intensity and task) on tonic and phasic heart rate and skin conductance responses were studied. 24 levelers and 24 sharpeners received a series of 25 auditory stimuli under two instructions (task): in one task subject had to listen carefully to possible small differences between tones (intake task) and in the other task the subject had to count back silently and could neglect the tones (rejection task). One third of each group received tones of 50 dB, one third 75 dB and one third 100 dB. Results show that task has a strong effect on tonic responses in the direction predicted from Lacey's intake--rejection hypothesis. There is hardly any effect of task on phasic responses: only at the first trial heart rate tends to decelerate more in the intake than in the rejection task. Stimulus intensity does not influence tonic responses but very strongly affects phasic responses: deceleration to 50 and 75 dB, acceleration to 100 dB. GSR amplitude increases monotonically as a function of stimulus intensity. There is no difference between levelers and sharpeners with regard to tonic responses. Concerning phasic heart rate, sharpeners decelerate more than levelers and levelers have greater GSRs than sharpeners. It is suggested that tonic changes are relatively more under cognitive control, whereas phasic changes are more of a non-cognitive reflex-like nature. A relationship between the distinction arousal-activation as proposed by Pribram and McGuinness (1975) and the results of this experiment was suggested.


Bilateral EEG differentiation of stimuli. The EEG of both right and left hemispheres was monitored while subjects were presented with words, music, arithmetical problems and abstract pictures, (15 trials of each treatment, 9 sec per trial). The left side of the brain was most activated during presentation of words and arithmetic, whilst the right side of the brain was most activated during presentation of music. In addition, the right side was more activated during exposure to arithmetic than to words. The pictures were not differentiated by either side of the brain and it is possible that they had the effect of deactivating or relaxing subjects, since they yielded high abundance values compared with other stimuli. This study confirms earlier findings showing significant between-hemisphere ratios for arithmetic and words. In addition, however, the within-hemisphere analyses show that music affects the right hemisphere (without inducing between-hemisphere changes) and that arithmetic also activates the right hemisphere if compared with the non-activating effects of words.


Signal onset and task variables in auditory evoked potentials. Two auditory evoked potential studies were run to determine the effects of stimulus rise times on amplitude of N1-P2 onset and offset potentials. Onset potentials were reduced with increased onset durations. No consistent relationship was found in N1-P2 offset potentials as a function of stimulus rise time. The effects of listening tasks and stimulus duration were also examined.


A modification of the Johnson-Neyman technique comparing two regressions, applied to treatment effects dependent on baseline levels. Treatment effects can often depend on baseline (pre-treatment) variables. This is ignored in many studies, or else the relationship is removed statistically by analysis of covariance. The latter method, however, assumes that slopes are equal and also loses information on the base-line contribution. Instead, the Johnson-Neyman technique does not assume that slopes are equal and furthermore permits examination of the baseline contribution. The method was devised for education research and is here extended to biological studies. However, the method assumes that variances are equal for the regressions, which might not be always so. The mmodification proposed accommodates inequalities of variance, whether intrinsic to the regressions or resulting from differences in group size. The Johnson--Neyman technique is discussed in relation to alternative analyses and, in appropriate situations, is considered to yield more information. Furthermore, with the refinement described, it involves even fewer assumptions and becomes more powerful.


Telemetry in health care.

Telemetry in health care. This article reviews the use of telemetry in health care. Monitoring of biological signals of medical importance such as the EKG, EEG, and EOG are described. Employment of telemetry in critical medical and surgical environs such as the delivery and operating suites is covered. The enormous importance of telemetry compatible with public communication systems such as voice grade telephone lines for remote medical care is discussed. The article does not enter into strictly engineering systems aspects in any detail; this was not the intent of the author. Nor is there any pretence to a completeness obviously not possible within its confines. It does concentrate on radio (wireless) telemetry transmissions over relatively great distances with freedom of patient movement.


Coating evolution with an implantable biological battery. A cathode of smooth platinum in a right endoatrial position and an anode of Domal magnesium were used to construct a hybrid bioelectric battery designed to power a pacemaker. In use the cathode is gradually covered by a coating consisting of a crystalline layer and a layer of connective tissue. This coating is responsible for the system change in the reduction process at the cathode level: the transfer from an oxygen to a hydrogen system. This system change causes a voltage drop in the bioelectric battery. This paper deals with oxygen diffusion across the tissue layer, as well as with various spectral and chemical analyses. A high level of calcium and phosphorus in the different analyses carried out seems to show that the presence of these elements is related to the coating of the cathode. The effect on the formation of the coating of the position of the cathode is also discussed.


Knee forces during the activity of getting out of a chair with and without the aid of arms. This is a preliminary study of the knee forces during the activity of getting out of a chair with and without the aid of arms. The equipment limited the study to the phase of the activity where no contact occurred between the subject and the chair seat. The model of the knee considered was two-dimensional, and quasi-static. Analysis showed that the forces acting in both compartments of the knee were comparable with those occurring during other non-strenuous activities such as level walking. It was also shown that the use of arms to aid rising from a chair causes considerable reduction in both joint and muscle forces.


Stochastic model of metastases formation. A mathematical model is developed to describe the dynamics of the hematogenous metastatic process to the lung from a solid tumor growing in a peripheral body site. The accumulation of tumor cell clumps of various sizes in the pulmonary circulation and the formation of metastatic foci are described by a non-homogeneous, two-dimensional Markov process. An analytical solution is found for the special case of metastases produced by the intravenous injection of tumor cell clumps. The system is decoupled experimentally to determine the time-varying entrance rate of tumor cell clumps into the circulation from a growing fibrosarcoma and the number of metastatic foci produced by the intravenous injection of tumor clumps. Model validation is based on comparisons of model simulations with data for the development of metastatic foci and the probability of cure following tumor excision. The model is used to simulate hypothetical therapy to prevent tumor metastases.


A stochastic model for censored-survival data in the presence of an auxiliary variable. In clinical trials and other investigations of survival time, information is often available on a time-dependent event other than survival. An example of such an auxiliary event in cancer studies is objective progression of disease. While some patients expire without experiencing objective disease progression, others die after progression is observed. This paper proposes a stochastic model which utilizes this type of information in the evaluation of survival time. Our intentions in presenting this model are to provide a means of relating survival and another time-dependent event to one another (each of which may be used in the evaluation of a patient's condition), and to obtain more precise estimates of survival time by exploiting its relationship with this other event. The intrinsic aspects of the model are related to the semi-Markov model proposed by Weiss and Zelen [1965]. An important difference is that the present model incorporates incomplete (censored) observations as well as covariante variables. Analysis of the model via the method of maximum likelihood and its testability are discussed. The methods are applied to the results of a recent lung cancer study.


A generalization of the one-sided two-sample Kolmogorov-Smirnov statistic for evaluating diagnostic tests. Suppose a continuous diagnostic measurement is used to classify patients, and suppose E1 false negative errors and E2 false positive errors result. The quantities E1 and E2, and the total number of misclassifications, L = E1 + E2, depend on the choice of cut-off value. We have determined the null distribution of min L, where minimization is over all possible cut-off values. The statistic, min L, can be used as a quick one-sided two-sample test, and min L is also useful for evaluating publications which present only a 2 X 2 table of false positives, false negatives, true positives and true negatives. In such cases, one can use min L to assess the usefulness of the diagnostic measurement, even if one suspects that the authors chose that particular cut-off value which minimized L after looking at the data. We extend these results to a more general weighted loss L = vE1 + MUE2 where v and mu are positive integers, and we show that min L is a generalization of the one-sided two-sample Kolmogorov-Smirnov statistic, and, indeed, exactly equivalent to that statistic for appropriate choices of v and mu.


Life tables with concomitant information. A regression model for the analysis of survival data adjusting for concomitant information is developed. The model presented can lead to the log linear exponential model (Glasser [1967]) and the life table regression model of Cox [1972]. In addition, the model described can be used to analyze data from the commonly employed actuarial life table. A discussion of the special case where one is comparing two survival curves is presented. The methods developed are illustrated using data from a clinical trial investigating treatments for lung cancer.


Use of the logistic model in retrospective studies. A logistic regression model is used to study the association between a dichotomous exposure variable and a disease. The method takes into account factors that may confound the association and leads to a quantitative study of the influence of factors which are related to the strength of the association. Special results are given for matched pair data. A case-control study relating post-menopausal estrogen use and endometrial cancer provides illustration.


The efficiency of the multiple trial randomized response technique. It has been proven theoretically that repeated administration of two-related-question randomized response technique (RRT) substantially increases the efficiency by reducing the variance of the estimate. The realtive efficiency of RRT to direct question with various number of trials per respondent under various degrees of truthful response and various conditions has also been examined. However, if the respondent becomes more concerned about his anonymity, then the mean square error may be larger. A field study of limited scope conducted in Taichung, Taiwan indicated that this multiple trial RRT model is at least as feasible as, if not more feasible than, the conventional single trial RRT. There was no sign suggesting cooperation of respondents had deteriorated because each was asked to repeat the procedures three times. If the number of trials did not exceed three, we believe that the increase in the cost of survey and "exhaustion" of respondents from repeated administration of the same set of questions would be negligible. Hence, we believe that this multiple trial technique is a feasible and useful survey method for study of various contemporary social problems of sensitive nature. More field testing, particularly in different socio-cultural settings, is necessary however, before a general conclusion may be drawn.


Comparing the joint agreement of several raters with another rater. In laboratory proficiency surveys which use reference laboratories for the evaluation of participant laboratories, a measure of the agreement of the participant laboratory with the reference laboratories is needed which considers the extent of agreement (or disagreement) among the reference laboratories themselves. In, a measure of nominal scale agreement, is proposed. In is interpreted as follows: Let a specimen be selected at random and rated by a reference laboratory which itself has been randomly selected from the n reference laboratories. If the specimen was also rated by the participant laboratory, this second rating would agree with the first at a rate In of the rate that would be obtained by a second randomly selected reference laboratory. An approximate (large sample) confidence interval for the ratio In is developed. In order to account for the more general case of scaled agreement, a weighted index of agreement is also considered.


Testing for a single outlier from a general linear regression. Several authors have considered the problem of detection of outliers from the general linear model Y = Xbeta + mu. Ellenberg [1973] among others, has advocated use of a detection method which involves examination of the set of internally standardized least squares residuals. Mickey [1974] and Snedecor and Cochran [1968], apparently concerned about the usefulness of an outlier detection method which is based on residual estimates that themselves are biassed by the presence of the outlier, have proposed two other alternatives. It is shown that the three approaches are exactly equivalent. A detection procedure is described which uses as its test statistic the maximum of the internally standardized least squares residuals, and upper and lower bounds for the percentage points of the test statistic are given by Bonferroni inequalities. The computations required to obtain these approximate percentage points are illustrated in a numerical example. Finally, a brief simulation study of the performance of the procedure illustrates that the power of the test can be influenced by the position of the outlier vis-a-vis the structure of the design matrix X.


Properties of diagnostic data distributions. In applications of statistical methods to medical diagnosis, information on patients' diseases and symptoms is collected and the resulting data-base is used to diagnose new patients. The data-structure is complicated by a number of factors, two of which are examined here: selection bias and unstable population. Under reasonable conditions, no correction for selection bias is required when assessing probabilities for diseases based on symptom information, and it is suggested that these "diagnostic distributions" should form the principal object of study. Transformation of these distributions under changing population structure is considered and shown to take on a simple form in many situations. It is argued that the prevailing paradigm of diagnostic statistics, which concentrates on incidence of symptoms for given disease, is largely inappropriate and should be replaced by an emphasis on diagnostic distributions. The generalized logistic model is seen to fit naturally into the new framework.


Skewness in commingled distributions.

Skewness in commingled distributions. A likelihood ratio test is given for distinguishing skewness from commingled distributions, using a power transform to remove skewness appropriately for each of the alternatives tested. The alternative hypotheses postulate that the transformed data are from one normal or a mixture of two or three normal homoscedastic distributions. Since each mixture has unique asymmetry, skewness is estimated simultaneously with the means, proportions and variance of components. Commingling cannot be rigorously proven in this way, as some other transform may provide a better approximation to normality. However, the error of asserting admixture whenever there is skewness has been avoided, and estimates of admixture parameters provide a basis for more conclusive tests in relatives or other populations. Two examples are given, one in which adjustment for skeweness left evidence of commingling.


Active immunological enhancement of sarcoma 180 in splenectomized Swiss mice. Maximal enhancement of tumor growth was obtained in Swiss mice, with or without spleen, inoculated with S180 within a subcutaneously implanted glass cylinder and pretreated with soluble tumor antigen.


Transient antibodies to DNA polymerase in acute hepatitis B and related diseases. Sera from patients with acute hepatitis, cirrhosis or chronic hepatitis, as well as sera from healthy carriers and controls were examined for HBS antigen, DNA polymerase activity and for antibodies to HBS, HBC and DNA polymerase. The data presented suggest that in acute hepatitis the DNA polymerase test enabled us to diagnose at least 20% more cases of hepatitis B than with the RIA but that the DNA polymerase test is of little value for the screening of blood donors since all the healthy carriers gave negative results. As concerns the antibodies to DNA polymerase they appeared in at least 50% of the patients with acute hepatitis, they were transient and only detectable at the early beginning of the disease. These antibodies were also found to be different from the anti-HBS and anti-HBC antibodies.


Interactions of short chain aliphatic hydrocarbons with human blood and haemoglobin A solutions. The solubilities  of methane, propane, butane and propylene were measured in human whole blood, human plasma and defatted haemoglobin solutions at 37 degrees C. For these four hydrocarbons the solubility coefficient (alpha = ml of gas STPD dissolved in one mal of the solvent under a pressure of one atmosphere) is higher in plasma than in an isoosmolar aqueous solution and higher in whole blood than in plasma. This is due to the presence of proteins and particularly of haemoglobin as shown by the dependence of alpha upon the concentration of haemoglobin in the medium. O2 capacity of human blood is not altered in the presence of these hydrocarbons and its affinity for O2 is slightly decreased.


Intestinal colonization with lactobacilli strains in neutropenic patients. In order to protect granulopenic patients against infections by pathogens from their own digestive tract, large amounts of lactobacilli were given to 5 patients whose intestinal flora had been suppressed by oral antibiotics. In all these cases, lactobacilli failed to prevent spontaneous recolonization of the gastrointestinal tract by enteric bacteria. Six attempts of colonization were made; in spite of the importance of the inoculum administered orally, large numbers of lactobacilli were recovered from stools in only two cases.


[Effect of cycloleucine on renal uptake of dibasic amino acids and cystine]. Cycloleucinead ministration (a synthetic amino acid) to rats produces a selective hyperaminoaciduria bearing on dibasic amino acids (lysine, arginine, ornithine) and cystine. Interference of cycloleucine with tubular reabsorption of these amino acids was studied "in vitro", using rat kidney cortex slices. When dibasic amino acids and cystine are at physiological concentrations, the data indicate that cycloleucine decreases their intracellular accumulation. However, effect of cycloleucine "in vitro" is not specific whatsoever its concentration; similar results are obtained with neutral amino acids such as alpha-alanine and valine. Inhibitory effects are not modified by the presence of several amino acids in the incubation medium. The results are interpreted as evidence for a dissociation between "in vitro" cellular accumu-lation and "in vivo" transepithelial transport.


An analysis of the effect of random and ageing mechanisms on the survival of platelets. Human platelets survival curves make it possible to estimate two factors which could be held responsible for the disappearance of cells: a) random destruction b) ageing of the platelets. This study was devoted to non-thrombocytopenic patients with chronic vascular disease. In 40% of the cases, the platelet destruction was found to be due to normal ageing of the cells combined with a random destruction process. The estimation of both of these parameters is of interest for the clinical survey of these patients. The statistical analysis of the remaining 60% of the cases presented here has demonstrated a pure linear disappearance of the labelled platelets. This obsveration favours ageing as the cause of the destruction of the platelets.


A simple method for the measurement of labelled compound incorporation into cells in culture. A simple method for the measurement of labelled compound incorporation into cells in layer culture was developed. Compared to other methods it proves to spare time and to be more sensitive owing to the fact that cells are not detached from the culture vials until the end of the manipulation as these are dissolved in the scintillation medium together with the cells just before counting.


Salivary immunoglobulin determinations: their diagnostic value in Sjögren's syndrome. Salivary immunoglobulins (IgA, IgG, IgM) determinations are performed on 74 patients with and without Sjögren's syndrome (SS). In normal subjects IgA is the only immunoglobulin detected in saliva by classical immunodiffusion methods. Correlations between the presence of IgG and/or IgM in saliva and other functional and objective clinical parameters and hsitological aspects of minor salivary glands are studied. In SS the prescence of IgG and/or IgM is in close relationship with "sialography index" which is the most reliable clinical investigation. These results point out that salivary immunoglobulins determinations constitute a simple and specific complementary test for SS diagnosis; they may allow the clinicians to observe the progress of the disease or to evaluate effectiveness of drugs.


An attempt of treatment of malignant melanoma with bovine pineal tissue. Seven incurable patients with disseminated malignant melanoma were injected (subcutaneously, sometimes intravenously) with homogenate of bovine pineal tissue. The daily injections of large quantities of pineal tissue appeared to be harmless for patients. Many-fold injections (during 9.5 months) of large quantities of pineal tissue (up to 3950 g) in 1 of 7 cases produced certain effect on subcutaneous metastases of malignant melanoma, i.e. causing reduction and even disappearance.


Significance of dimers to the size of newly synthesized DNA in UV-irradiated Chinese hamster ovary cells. DNA synthesized after UV irradiation is smaller than that in unirradiated cells even when pulse-labeling times are increased to compensate for the overall reduction in the rate of DNA replication. By isolating newly replicated DNA, incubating it with dimer-specific endonuclease from Micrococcus luteus, and analyzing it on alkaline sucrose gradients, we have been able to demonstrate that this DNA is synthesized in segments corresponding in size to the interdimer distance on the parental strand. In addition, the same DNA analyzed on neutral gradients shows no reduction in molecular weight as a result of UV irradiation and/or endonuclease digestion. Our data are thus inconsistent with the presence of "gaps" in newly synthesized DNA opposite the dimers on the parental strand. We suggest that if such gaps are produced as a result of delayed synthesis around dimers, they are filled before the growing point reaches the next dimer.


Chemotropism indices for polymorphonuclear leukocytes. Trajectories of polymorphonuclear leukocytes which are responding to a chemical gradient are analyzed in order to deduce probability distributions of the angles between successive path segments. The turn angle probability distributions thus obtained are seen to be strongly dependent on the direction of locomotion prior to a turn, in that cells usually turn to maintain alignment along an axis directed towards the chemoattractant source. A mathematical model based on these observations is developed in order to understand the relationship between net chemotactic response and parameters characterizing stochastic movements of individual cells. In particular, the manner in which the chemotropism index depends on details of the turn-angle distributions is examined. When bias in the direction of turn is induced by a chemotactic field, transition from random motion to directed response occurs most abruptly if the turn-angle distribution is narrow. "Accommodation," viz., a dependence of the mean angle of turn upon prior orientation, is found to have relatively little effect on the magnitude of the response.


Effects of light adaptation on the purple membrane structure of Halobacterium halobium. Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.


On electron transfer.

On electron transfer. The tunneling transfer of electrons between two sites, appropriate to biological intermolecular electron transfer, contains physical considerations which are not important in the tunneling transfer between two metals. Analyses (such as the recent one by Hales) based on the well-known formula for the latter but applied to the former case are quantitatively and qualitatively misleading.


Prediction of the conformation of the histones. The secondary structures of the histones, H1, H2A, H2B, H3, and H4 have been predicted utilizing the predictive scheme of Chou and Fasman (Biochemistry 13:211, 222[1974]) and a new set of conformational parameters based on the X-ray data of 29 protein structures. The alpha-helical, beta-sheet, reverse beta-turns, and random coil regions of these proteins are carefully delineated. Structures are specified which are most probably under various environmental conditions, i.e., for changes in ionic strength, association between histones and in association with DNA. Potential conformational changes within these histones are also predicted.


Fragmentation of chromatin with 125I radioactive disintegrations. The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.


Computer simulation of movement-generating cross-bridges. A stochastic computational method was developed to study properties of cross-bridge models for muscle contraction, by following the time history of individual cross-bridge model of Andrew Huxley (1957) and a modified two-state model with more realistic behavior during steady stretching are used as examples. The method can readily compute steady-state force during shortening and stretching and force-transients following rapid changes in length. Computations of velocity with a steady load and of velocity transients are more sensitive to the randomness inherent in the stochastic method.


Computer simulation of flagellar movement. IV. Properties of an oscillatory two-state cross-bridge model. A stochastic computational method is used to examine the properties of a simple two-state cross-bridge model, of a type which has been shown previously to self-oscillate without requiring any feedback control of the active process. The force transients obtained with this model show the major features observed with oscillatory insect fibrillar flight muscle. The effects of viscosity and cross-bridge detachment rate on the frequency of oscillation of the model resemble the effects of viscosity and ATP concentration on flagellar oscillation, and the relationship between turnover rate and frequency of oscillation is also consistent with observations on flagella. However, the amplitude of oscillation of the model does not show the degree of frequency-independence which is typical of flagella.


Nuclear magnetic resonance measurement of skeletal muscle: anisotrophy of the diffusion coefficient of the intracellular water. The anisotropy of the spin-diffusion coefficient Ds of water protons in skeletal muscle has been studied by pulsed NMR methods. The mid-portion of the tibialis anterior muscle of mature male rats was placed in a special sample holder by means of which the muscle fiber orientation theta relative to the diffusion direction could be varied over the range 0 degrees less than or equal to theta less than or equal to 90 degrees. The value of Ds(theta) was determined for theta = 0 degrees, 45 degrees, and 90 degrees. The measured anisotropy Ds(0)/Ds(90) was 1.39, and the value of Ds(0) was 1.39 X 10(-5) cm2/s. These results are interpreted within the framework of a model calculation in which the diffusion equation is solved for a regular hexagonal network similar to the actin-myosin filament network. The large anisotropy, and the large reduction in the value of Ds measured parallel to the filament axes lead to two major conclusions: (a) interpretations in which the reduction in Ds is ascribed to the effect of geometrical obstructions on the diffusion of "free" water are ruled out; and, (b) there is a large fraction of the cellular water associated with the proteins in such a way that its diffusion coefficient is substantially reduced.


Relaxation phenomena in human erythrocyte suspensions. Previous work has shown that the application of the Joule heating temperature jump technique of Eigen and de Maeyer to an istonic suspension of human erythrocytes induced an interiorization of [3H-A1glucose and a hemolysis of the red cells (Tsong, T.Y., and E. Kingsley, J. Biol. Chem. 250:786 [1975]). The result was interpreted as due to the thermal osmosis effect. Further considerations of the various effects of the Joule heating technique indicate that the hemolysis of the red cells may also be caused by the rapid dielectric perturbation of the cell membranes. By means of turbidity measurements of the suspensions we have detected at least four relaxation times. Two of the faster ones (tau1 approximately 20 mus and tau2 approximately 5 ms) are tentatively attributed to water relaxations in the membrane structures. The other two are attributed to membrane ruptures (tlag approximately 0.1s) and the hemolysis reaction (tau3 approximately 0.5 s). Studies with the erythrocytes from different hematological disorders indicate that whereas the two slower relaxations are sensitive to the overall physical property of the red cell membranes the two faster relaxations are not. These observations are consistent with the above assignment of the relaxation processes. The apparent activation energies are, above assignment of the relaxation processes. The apparent activation energies are, respectively, 8.4, 12.0, and 11.8 kcal/mol for the tau1, tau2, and tau3 reactions. Experiments with erythrocyte ghosts indicate a single relaxation for the water permeation, and biphasic kinetics for the membrane rupture and resealing reactions. The phenomena reported here may contribute to our understanding of water transport and molecular release in cellular systems.


Models for 1/f noise in nerve membranes. A recently proposed model for 1/f(w-1) noise in nerve membrane (Clay and Schlesinger, 1976; Lundström and McQueen, 1974) is shown to be mathematically inconsistent in several respects. A self-consistent model based on similar membranes lipid orientation fluctuation effects is proposed.


Branched bimolecular lipid membranes.

Branched bimolecular lipid membranes. Branched bimolecular lipid membranes separating three and four aqueous phases have been formed. The procedure is based on the technique of Montal and Mueller generalized to three and four lipid surface films spanning an appropriate aperture. The technique to produce Teflon structures for the mechanical support of branched bilayers is presented. The existence of the branched bilayer was established by measuring the specific capacity, specific resistance, and the gramicidin-induced single channel conductance of each branch. These structures should facilitate the study of transport properties of ionophores and other molecules and may also serve as model systems for the study of cell fusion.


Laser light-scattering spectroscopy: a new application in the study of ciliary activity. A uniquely precise and simple method to study ciliary activity by laser light-scattering spectroscopy has been developed and validated. A concurrent study of the effect of Ca2+ on ciliary activity in vitro by laser scattering spectroscopy and high speed cinematography has demonstrated that this new method is simpler and as accurate and reproducible as the high speed film technique.


Exact solution of the unidimensional Poisson-Boltzmann equation for a 1:2 (2:1) electrolyte. The unidimensional Poisson-Boltzmann equation for a 1:2 (2:1) electrolyte has been solved analytically. The results have been compared with those obtained from the linearized equation. It is shown that in physiological conditions the difference may be greater than 10%. The value of the derivative of the potential in x=0, (dpsi/dx)x=0, has been used by many authors in the evaluation of the superficial charges of biological membranes. The value of (dpsi/dx)x-0 have also been compared with the ones derived from the linearized equation. The difference may be greater than 25%. Our results suggest that the linearization of the Poisson-Boltzmann equation for a 1:2(2:1) electrolyte may be greatly misleading.


Activation-inactivation coupling in Myxicola giant axons injected with tetraethylammonium. Myxicola giant axons internally injected with tetraethylammonium chloride to block potassium currents were examined under voltage clamp. The sodium inactivation time constants obtained from the decline in INa during step depolarizations were substantially smaller than those obtained using conditioning prepulses to the same potentials and the ratios agreed with previous observations using TTX. Inactivation shifts were also measured and found to be comparable to previous results.


The electrical resistivity of cytoplasm. The apparent cytoplasmic resistivity of two different giant cells has been measured using an extension of a previously developed single microelectrode technique. Each cell is penetrated by a metal microelectrode whose complex impedance is measured as a function of frequency between 500 kHz and 5.7 MHz. By plotting the measured impedance data on the complex Z plane and extrapolating the data to infinite frequency, the substantial effects of electrode polarization can be overcome. For Aplysia giant neurons and muscle fibers of the giant barnacle, the extrapolated cytoplasmic specific resistivities are 40 and 74 omega-cm, respectively, at infinite frequency. The barnacle data are in excellent agreement with sarcoplasmic resistivity values derived from the measured cable properties of other marine organisms, and from high frequency conductivity cell measurements in intact barnacle muscle tissue. In the Aplysia neurons, the frequency-dependent part of the electrode impedance is larger when the electrode is in a cell than when it is in an electrolyte solution with the same specific resistivity as the aqueous cytoplasm; however, the phase angle of the frequency-dependent component of the electrode impedance is the same in both cases. This suggests that the high apparent values of cytoplasmic resistivity found using the single microelectrode technique at lower frequencies probably reflect an artifact caused by reduction of the effective surface area of the electrode by intracellular membranes, with a corresponding increase in its polarization impedance.


Volume of reaction by the Archibald ultracentrifuge method (lobster hemocyanin). Samples of lobster hemocyanin (Homarus americanus) under conditions of reversible reaction between whole (25 S) and half (17 S) molecules have been subjected to accurately known nitrogen pressures in analytical ultracentrifuge cells. A modified pressurization chamber of the type developed by Schumaker and colleagues has been constructed for this purpose. The molecular weight was then determined at the top (liquid-gas) meniscus, by means of the Archibald method. The logarithmic dependence upon pressure of the derived equilibrium constant then gave directly the volume of reaction. Experiments were performed in veronal-citrate buffers at pH 8, where the molar volume of formation of whole (dodecameric) molecules from half molecules appears to be negative, and at pH 8.46 in veronal-citrate buffer in the presence of 0.003 molar free calcium ion, where the molar volume of formation was estimated to be + 390 cm3/mole. In glycine-sodium hydroxide buffer at pH 9.6 containing 0.0047 molar free calcium, the molar volume of formation of whole molecules was estimated to be +120 +/- 70 cm3, corresponding to an estimated difference in partial specific volume between whole molecules and half molecules of only 1.3 (10)-4cm3/gram. The correctness of the sign of this value in glycine buffer has been verified by pressure-jump light-scattering experiments.


Simulation studies of self-associating systems; discrimination between specific and isodesmic associations. The possibilities of discriminating between definite and indefinite (isodesmic) modes of self-association are explored by fitting the different models to stimulated data, using non-linear least-squares curve-fitting to determine the fitting parameters for real and impostor models. It was found that over an extensive range of values for the equilibrium constant of a non-ideal isodesmic generating model, only a non-ideal monomer-dimer-tetramer-octamer was a successful impostor model. Some criteria for rejecting inappropriate models are discussed.


Sedimentation equilibrium techniques: multiple speed analyses and an overspeed procedure. The use of a single high-speed sedimentation equilibrium experiment to analyze mixed associating systems is inadequate to determine the association mode even if the molecular weight of one species is known. Simultaneous analysis of the concentration distributions at three equilibrium speeds greatly reduces the lack of uniqueness. Linear least-squares multi-speed fits discriminate between association models in which the molecular weights are assumed. Experiments at a series of initial concentrations as well as rotor speeds further increases the discrimination. An overspeed procedure is proposed. The overspeed time depends only slightly on the sedimenting species molecular weight, but significantly on the frictional ratio. A minimal overspeed time may be estimated.


Improvement in precision of sedimentation-equilibrium experiments with an on-line absorption scanner. An on-line computerized optical absorption scanner for the analytical ultracentrifuge has been developed and tested. It makes direct use of the instantaneous photomultiplier output, which is digitized and averaged by the computer. It incorporates a stepping motor to drive the photomultiplier, two analog-to-digital converters, and a device which monitors precessional movement of the rotor. The scanner has been employed chiefly in sedimentation equilibrium experiments, and techniques have been devised to correct for the adverse effects of inhomogeneities of apparent absorbance which arise from the cell windows. Correction for rotor movements which occur during the scan is also possible. The completed apparatus is a successful one, and provides a significant improvement in precision, speed and convenience over the commercially available scanners. The limitations on precision inherent in the present design are examined, and it is concluded that the combined effects of window inhomogeneities and rotor movement lead to uncertainty of the measured absorbance which is unlikely to be reduced below +/- 0.001 A.


Molecular weights and molecular-weight distributions from ultracentrifugation of nonideal solutions. Ultracentrifugation, membrane osmometry and capillary viscometry experiments have been performed on two dextran samples, which have molecular-weight distributions (MWDs) similar to those of dextrans used as blood plasma extenders. The manufacturer reported values of Mn and MW, determined by end group analysis and by light scattering, respectively. Our values of Mn, determined by osmometry, and MW, calculated from ultracentrifugal and viscometry experiments, agreed quite well with the manufacturer's results. Good agreement was obtained with values of MW and BLS (the light scattering second virial coefficient) obtained from sedimentation equilibrium experiments at different speeds using sector or nonsector-shaped centerpieces. Several ways of obtaining MW, MZ and BLS from sedimentation equilibrium experiments are presented. We have also shown how to obtain the speed-dependent term of the sedimentation equilibrium second virial coefficient. Both BLS and the speed-dependent nonideal terms could be used to correct the sedimentation equilibrium data, so that ideal values of d in c/d(r2) or dc/d(r2) could be estimated and used to obtain the MWDs of the dextran samples. Both Donnelly's and Scholte's methods were used with the sedimentation equilibrium data. With both methods, unimodal MWDs were encountered, which gave good agreement with the manufacturer's MWDs, obtained by a combination of analytical gel chromatography and light scattering. Uncorrected sedimentation equilibrium data gave MWDs quite different from the manufacturer's results. The MWD calculated from the differential distribution of sedimentation coefficients also gave a unimodal MWD, but this MWD did not give a good agreement with the sedimentation equilibrium results or with the manufacturer's results.


Sedimentation in the ultracentrifuge and diffusion of macromolecules carrying electrical charges. The calculation of Alexandrowicz and Daniel of the charge effect in the sedimentation of macromolecules carrying electrical charges was reformulated by introducing buoyant molecular weights and concentrations of electroneutral components only (rather than those of the ionized species) in the final equations. Some interesting limiting cases are discussed. A similar calculation was performed for the case of isothermal diffusion. It is also shown how the present discussion relates to more elaborate treatments of the sedimentation and diffusion of polyelectrolytes by the methods of irreversible thermodynamics.


Numerical study of the Johnston-Ogston effect in two-component systems. Numerical solutions of the Lamm equation are presented for systems exhibiting the Johnston--Ogston effect. From these solutions it is apparent that the movement of the maxima of the concentration gradient curves reflects the sedimentation velocity of the slow or fast components in their appropriate plateaus. A simple generalization of the Johnston--Ogston analysis is presented, valid for all centrifugation times in a radial field and sector shaped cell provided only that there exist both a plateau of the slow component by itself and the mixed plateau with both slow and fast components present.


Anomalies in sedimentation. V. Chains at high fields, practical consequences. The theory of a preceding paper [B.H. Zimm, Biophys, Chem. 1 (1974) 279] is used to calculate a numerical table for the change of sedimentation coefficient with centrifugal field for chain molecules. A simple formula is found to fit the results within 1.3% up to the centrifugal field at which S/So = 0.377; this formula is S/So=(1+0.1155y2)-1/4, where y is proportional to M2/So times the centripetal acceleration, M being the molecular weight and So the sedimentation coefficient at zero acceleration. Applying this formula to DNA, we conclude that at a given centrifuge speed the sedimentation coefficient must reach a maximum at a particular molecular weight and be smaller at higher molecular weights. The value of the maximum depends on the conditions, but can come at less than 150 S for DNA under typical conditions. When a maximum is present, the profile of a sedimenting non-homogeneous band is also severely distorted.


UV laser scanning and fluorescence monitoring of analytical ultracentrifugation with an on-line computer system. A new optical system for the analytical ultracentrifuge is described, which permits sedimentation to be monitored by fluorescence. The optical system is based on a laser light source, which is focused to a narrow (50 micron) beam. The radical scanning of the beam provides information on the distribution of fluorescing material with distance in the centrifuge cell. Data collection and processing are performed in conjunction with an on-line computer system which sorts incoming fluorescence pulses according to rotor hole and cell sector, averages families of pulses to improve signal to noise ratios and fits the data (in the experiments reported here) to equations to determine sedimentation coefficients. Initial experiments with the system have been performed with bovine serum albumin and indicate that sedimentation can be readily monitored by fluorescence with solutions at concentrations as low as 20 micrograms per ml, with excitation at 257 nm. At these concentrations, the optical density is only in the 0.01 range, too low for experiments with absorption-scanner optical system. Even lower concentrations can be used when fluorescent labels are used with excitation in the visible region of the spectrum. The preliminary studies indicate that fluorescence monitoring of sedimentation will substantially enhance the range of experimental possibilities in ultracentrifugation by improving both the sensitivity of measurements and the discrimination between sedimenting species on the basis of their fluorescence characteristics.