Proton nuclear magnetic resonance studies of hemoglobin M Milwaukee and their implications concerning the mechanism of cooperative oxygenation of hemoglobin.

Hemoglobin M Milwaukee (beta67E11 Val leads to Glu) is a naturally occurring valency hybrid containing two permanently oxidized hemes on the beta chains. In this mutant, the two abnormal beta chains cannot combine with ligands whereas the two alpha chains are normal and can combine with oxygen with a Hill coefficient varying from 1.1 to 1.3 [Udem et al. (1970), J Mol. Biol. 48, 489]. High-resolution proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the exchangeable, ring-current shifted, ferrous and ferric hyperfine shifted resonances of Hb M Milwaukee in the absence and presence of organic phosphates. The alpha-heme environment, as manifested by the ring-current shifted resonances in the liganded form as well as the ferrous hyperfine shifted resonances in unliganded form, and subunit interactions, as manifested by the exchangeable resonances, are similar in Hb M Milwaukee to those in normal adult human hemoglobin. Organic phosphates can partially or completely inhibit the structural transformation which normally accompanies the binding of oxygen or carbon monoxide to Hb M Milwaukee. Upon stepwise addition of oxygen to deoxy Hb M Milwaukee, the hyperfine shifted resonance spectra of ferric beta chains show features which cannot be attributed to either fully deoxy or oxy species. However, the spectra for partially oxygenated Hb M Milwaukee can be described as an appropriately weighted average of the spectra of sero, singly, and doubly oxygenated species. The ferric hyperfine shifted resonance spectrum of the singly oxygenated intermediate has been calculated by a method employing least-squares analysis of the spectra of partially oxygenated Hb M Milwaukee at several values of oxygen saturation. The spectrum of this intermediate exhibits features which cannot be accounted for by a two-structure model. The present results are consistent with a sequential model for the oxygenation of this mutant hemoglobin. In view of the similarities between normal adult hemoglobin and Hb M Milwaukee, it is suggested that a two-state concerted allosteric model does not provide an adequate description of the structure-function relationship in normal adult hemoglobin.


Excision of 7-bromomethylbenz[a]anthracene--DNA adducts in replicating mammalian cells.

The excision of 7-bromomethylbenz[a]anthracene--DNA adducts was studied in two cell lines (HeLa S-3 and Chinese hamster V-79379A). In both cell lines, carcinogen-modified adenine residues were excised more readily than the modified guanine residues and the percentage of the total products excised decreased after treatment with higher concentrations of carcinogen. At the highest concentrations used in the Chinese hamster cells, neither DNA synthesis nor excision was detected. The lowest concentration used for these cells permitted almost 100% survival and all the DNA was replicated in a 30-h interval even though 50% of the initial damage was still present. The two- to threefold lower sensitivity of the Chinese hamster cells (compared with the Hela cells) to the carcinogen is attributed to this capacity for replication of DNA on a damaged template since the two cell lines' capacities for excision of the chemical damage were found to be comparable.


Dissociation of the 7S-nerve growth factor complex in solution.

Sedimentation and gel-filtration studies of mouse submandibular gland 7S-nerve growth factor (NGF) reveal that this complex dissociates to yield its components at concentrations much higher than those required to exhibit biological activity. Results further indicate that the alpha and gamma protein c omponents of the 7S-NGF complex probably play no role in its biological activity when tested in vitro. The dissociation behavior of 7S-NGF is quite different from the properties of very dilute solutions of the NGF secreted by mouse L cells and of that present in fresh, unpurified submandibular gland homogenates, since both of these proteins display high molecular weights at concentrations where 7s-NGF is fully dissociated. Thus, it could be that 7S-NGF is not the form in which NGF exists in the mouse submandibular gland.


Intensity effects on the fluorescence of in vivo chlorophyll.

A technique for measuring relative quantum yields of fluorescence with a picosecond streak camera is described. We show that Chlorella pyrenoidosa exhibit an intensity dependent quantum yield when irradiated with single picosecond light pulses. This effect also occurs under conditions that inhibit the activity of the reaction centres, which can therefore be excluded as the cause. When a pulse train (pulse separation 6.9 ns) was used, the quantum yield was further reduced by the light absorbed from previous pulses, which indicates the formation of a quenching species having a relatively long lifetime. Absolute quantum yields calculated from the fluorescence decay show that single excitation pulses of 3 - 10(13) photons/cm2 give results comparable to those obtained by very low intensity methods.


The effect of dihydroxyacetone phosphate and 3-phosphoglycerate on O2 evolution and on the levels of ATP, ADP and Pi in isolated intact chloroplasts.

Addition of dihydroxyacetone phosphate (2.5 mM) or 3-phosphoglycerate (2.5 mM) to a suspension of isolated intact chloroplasts, which contains Pi only in low concentrations (0.2 mM) leads to a competitive inhibition of Pi uptake in the light. In consequence, the ATP/ADP ratio is strongly decreased. The rate of O2 evolution is also reduced under these conditions, but the degree of inhibition is much higher after addition of dihydroxyacetone phosphate than after addition of 3-phosphoglycerate. Therefore, besides the competitive inhibition of Pi uptake, additional effects of dihydroxyacetone phosphate and 3-phosphoglycerate on O2 evolution and CO2 fixation of isolated intact chloroplasts must occur, which are discussed.


Picosecond laser study of fluorescence lifetimes in spinach chloroplast photosytem I and photosystem II preparations.

Fractions enriched in either Photosystem I or Photosystem II have been prepared from chloroplasts with digitonin. A more detailed analysis of the decay kinetics of fluorescence excited by a picosecond laser pulse has been possible compared to experiments with unfractionated systems. The Photosystem I fractions show a very short component (less than or equal to 100 ps) at room temperature which is apparently independent of pulse intensity over the range of photon densities used (5 - 10(13)--1 - 10(16) photons cm-2). The Photosystem II fraction has a short initial lifetime at room temperature which is strongly intensity-dependent approaching 500 ps at low photon densities, but decreasing to close to 150 ps at the highest photon densities. All of these room temperature decays appear to be non-exponential, and may possibly be fitted by at t1/2 expression, expected from a random diffusion of excitations via Förster energy transfer. On cooling to 77K, lifetimes of both Photosystem I and Photosytem II increase, the lengthening with Photosystem I being more striking. The Photosystem I decays become intensity dependent like the Photosystem II, and at the lowest photon densities decays which are more nearly exponential within the experimental error give initial lifetimes of about 2 ns. The non-exponential decays seen at high photon densities appear to fit a t1/2 expression.


The role of plastoquinone and beta-carotene in the primary reaction of plant photosystem II.

Extraction of Triton Photosystem II chloroplast fragments with 0.2% methanol in hexane for 3 h results in the removal of 90 to 95% of the plastoquinone in the original preparation. The extracted fragments (chlorophyll:plastoquinone ratio, 900: 1) showed no P-680 photooxidation at 15 K after a single laser flash. The extracted fragments also showed no light-induced C-550 absorbance change at 77 K. Reconstitution of the primary reaction of Photosystem II, as evidenced by restoration of low-temperature photooxidation of P-680, could be obtained by the addition of plastoquinone A but not by the addition of beta-carotene. The addition of beta-carotene plus plastoquinone A restored the C-550 absorbance change. These results indicate that plastoquinone functions as the primary electron acceptor of Photosystem II and that beta-carotene does not play a direct role in the primary photochemistry but is required for the C-550 absorbance change.


Formation of electrical field accompanying temperature jump in isolated spinach chloroplasts.

Temperature-jump-induced absorbance changes of spinach chloroplasts in the dark were studied. After the temperature rise, a fast absorbance decrease and a succeeding slow absorbance increase were observed at the wavelength of 515 nm. The spectrum of the fast phase had positive maxima (increase in absorbance) at 430, 470 and 673 nm and a negative maxima (decrease in absorbance) at 525 nm. Permeant ions, tetraphenylboron-, tetraphenylarsonium+, and tetraphenylphosphonium+, decreased the extent of the fast absorbance change and increased the rate of slow recovery. Additions of inorganic potassium salts had a similar effect. Valinomycin, added in the presence of potassium ion, also increased the rate of slow recovery. These ions and ionophore had a parallel effect also on the recovery of flash-induced 515-nm absorbance change in chloroplasts. Electroneutral nigerericin did not affect the temperature-jump-induced absorbanc change. These results suggest the formation of electrical field across the thylakoid membrane in the dark accompanying the temperature rise. A possible involvement of the movement of water molecules (thermo-osmosis) in the observed absorbance changes is also discussed.


Electron transport between plastoquinone and chlorophyll Ai in chloroplasts. II. Reaction kinetics and the function of plastocyanin in situ.

The light-induced reaction kinetics of electron carriers between the two light reactions were studied in spinach chloroplasts. 1. The difference spectrum of the absorbance changes of plastocyanin in situ was separated from superimposing absorbance changes by flash titration described in the preceding paper (Haehnel, W. (1973) Biochim. Biophys. Acta 305, 618-631). Relative amounts of 2 : 1 electron equivalents were observed for plastocyanin and chlorophyll a1 (P-700). 2. A balance of the electron equivalents released from reduced plastoquinone and simultaneously accepted by oxidized plastocyanin, cytochrome f and chlorophyll a1 indicated a quantitative electron transfer. Additional electron carriers between plastoquinone and light reaction I can be excluded with an accuracy of about +/-0.3 electron equivalents per light reaction II. 3. The time course of the absorbance changes of plastocyanin was measured at 584 nm with negligible interference with other absorbance changes. The reduction kinetics show an initial lag followed by a rise with a half time of about 20 ms. The redox states of plastocyanin and chlorophyll a1 during this reduction via the rate-limiting step between the light reactions and during oxidation by weak far-red light suggest a true equilibrium constant of about 20. 4. The simultaneous oxidation and reduction kinetics of plastoquinone, cytochrome f, plastocyanin and chlorophyll a1 induced by two successive groups of saturating flashes after far-red illumination were measured. The oxidation kinetics of plastocyanin and the simultaneous reduction kinetics of chlorophyll a1 after the single flashes indicate a quantitative electron transfer with a half time of 200 mus. 5. The fast reduction of chlorophyll a1 by plastocyanin showed no effect of the inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or of reduced phenazine methosulfate. But it was completed inhibited after KCN incubation. 6. The oxidation kinetics of cytochrome f were reinvestigated with high time resolution from the difference of absorbance changes at 554 minus 540 nm to minimize the disturbing interference with other absorbance changes. Absorbance changes measured 554 nm alone do not reflect kinetics of cytochrome f. The half time of the oxidation was faster than 40 microns. 7. The observed reaction kinetics gave evidence for a function of cytochrome f between plastoquinone and chlorophyll a1 in parallel to plastocyanin. In addition, they indicate that the greater portion of linear electron transport passes plastocyanin. The complex interaction between cytochrome f and chlorophyll a1 is discussed.


The kinetic behavior of P-700 during the induction of photosynthesis in algae.

The kinetics of P-700 were examined spectrophotometrically during the induction of photosynthesis in algae. A pronounced oscillation was observed in the redox level of P-700 upon illumination of dark-adapted cells. The dark adaptation required approximately 1 min. The oscillation may be described as an initial rapid oxidation reaching a peak at approx. 50 ms followed by complete reduction of the pool of P-700. A subsequent slower oxidation resulted in attainment of the final state around 1 s. The main features of the oscillation were qualitatively the same in a wide variety of algae. The modulation in redox level of P-700 required high intensity activation of both photosystems and was eliminated by pre-illumination of the cells with weak short wavelength light but not by longer wavelengths absorbed primarily by Photosystem I. We propose that the P-700 modulation is directly related to the fast redox changes in Photosystem II which occur during the induction of photosynthesis. Cells incubated with methyl viologen did not show the P-700 oscillation confirming the suggestion previously advanced that exhaustion of Photosystem I acceptor and kinetic limitations in the carbon reduction cycle partially control the fast phase of photosynthetic induction.


Appearance and composition of chlorophyll-protein complexes I and II during chloroplast membrane biogenesis in Chlamydomonas reinhardi y-1.

The chlorophyll-protein complexes I and II have been isolated and anlyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during greening and degreening of Chlamydomonas reinhardi y-1. At all stages of membrane formation, the complexes, when present, have a constant composition. Chlorophyll-protein complex I consists of a major polypeptide(s) of molecular weight 64,000 synthesized in the chloroplast, to which about 29 chlorophyll a molecules are bound. The complex is not detected when other polypeptides of chloroplastic origin, related to both Photosystem I and Photosystem II activities, are not synthesized. However, Photosystem I activity can develop in membranes in which chlorophyll-protein complex I is not detectable. Chlorophyll-protein complex II consists of two polypeptides of cytoplasmic origin, molecular weights 24,000 and 22,000, which bind 12 chlorophylls (a and b). The chlorophyll-protein complex II can be detected in membranes in which the development of photosystem II activity is prevented. Clipping of a Mr = 2000 fragment(s) from the Mr = 22,000 polypeptide following trypsin digestion of membranes, does not affect the complex. The detection of the complexes is possible only in membranes in which the simultaneous synthesis of both the chlorophyll and the corresponding polypeptides occurs. The 28,000 dalton polypeptide, reported to be present in the chlorophyll-protein complex II, comigrates with the complex but apparently is not part of the complex itself. The apparent molecular weight of the chlorophyll-protein complexes I and II are 88,000 and 28,000, respectively. The minimal true value for complex I is 89,000 or 154,000 and for complex II is 56,000.


Chloroplast membrane sidedness. Location of plastocyanin determined by chemical modifiers.

Intact grana and stroma membranes (outer membrane absent) and detergent or sonication disrupted thylakoid membranes were treated with the hydrophilic covalent chemical modifiers [35S]diazonium benzene sulfonic acid ([35S]DABS) and [14C]glycine ethylester plus 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CDIS). Plastocyanin was purified using column chromatography followed by polyacrylamide gel electrophoresis and the incorporation of [35S]DABS and [14C]glycine ethylester into plastocyanin was determined by slicing the gels and counting the radioactivity in the plastocyanin band. Plastocyanin isolated from thylakoids disrupted prior to chemical modification binds two to four times as much of either modifier than the plastocyanin isolated from intact chloroplasts. This ratio is five to ten times lower than the ratio expected for a component buried behind the permeability barrier of a membrane. The data suggest that plastocyanin is partially exposed at the external surface of the thylakoid membrane rather than being completely buried in, or behind, the lipo-protein membrane.


Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation.

Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 degrees C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor tension developed upon transferring native or labelled skinned fibers from relaxing to rigor solutions lacking Ca2+ was very small but could be enhanced by progressively incresing Ca2 concentration; the rigor tension decreased with increasing sarcomer length. Polarization of fluorescence of skinned fibers reacted with 1,5-IAEDANS was measured along the line of excitation as well as at 90 degrees to it. The mean values of parallel and perpendicular components of polarization of labelled fibers measured at 0 degrees were close to the values obtained for native fibers irrigated with 1,5-IAEDANS-labelled heavy meromyosin fiber "ghosts" irrigated with labelled heavy meromyosin, and oriented bundles of myofibrils reacted with the same fluorophore. Skinned fibers stretched above the rest length and then irrigated with 1,5-IAEDANS-labelled heavy meromyosin gave rise to polarized fluorescence close to the values theoretically predicted for an assembly of helically arranged fluorophores. Using 90 degrees detecttion system a satisfactory fit to the theory could be obtained from single fibers labelled with 1,5-IAEDANS and measured in rigor. The angle between the fiber axis and the direction of the emission dipole of 1,5-IAEDANS attached to subfragment-1 was estimated to be near 40 degrees.


Uncoupling of mitochondrial respiration by ADP.

Even when oxidative phosphorylation is blocked completely by addition of high concentrations of oligomycin plus aurovertin, the addition of ADP to a suspension of mitochondria containing a high concentration of ATP inside the mitochondria induces a stimulation of respiration and oxidation of nicotinamide nucleotide. It is concluded that transport of ADP into mitochondria with a high endogenous ATP/ADP ratio requires energy.


The interaction of polyxanthylic acid with polyadenylic acid.

The stoichiometry of interaction between polyxanthylic acid (poly(X)) and polyadenylic acid (poly(A)) was investigated by construction of mixing curves as a function of wavelength. Wavelengths were found which exhibited a break at 48-50 mol % poly(A) or two breaks, one at 48-50 mol % poly(A) and the other at 33-30 mol % poly(A). The melting profile (in 0.1 M salt) of the 50 mol % poly(A) mixture was monophasic (Tm=83 degrees C) at all wavelengths but that of the 33 mol % poly(A) mixture was biphasic showing a transition at Tm=40 degrees C and another at Tm=83 degrees C. Construction of a mixing curve between poly(A) and poly(X) at a temperature of 52 degrees C gave rise to plots which showed only one break (at 49--50 mol % poly(A)) at all wavelengths. Thus, while (at 20 degrees C in 0.1 M salt, pH 7) both poly(A)-poly(X) and poly(A)-2 poly(X) form at their respective stoichiometric end-points, the triplex poly(A)-2 poly(X), upon melting undergoes a disproportionation reaction resulting in the loss of one poly(X) strand and the formation of the poly(A)-poly(X) duplex.


Peptidyl transferase activity in wheat germ ribosomes. Effect of some antibiotics.

The formation of N-acetyl-leucyl-puromycin in a "fragment reaction" catalyzed by 80 S ribosomes from wheat germ was characterized. The reaction product was identified by high-voltage electrophoresis. The fragment reaction is inhibited by sparsomycin, blasticidin S, gougerotin and to a lesser degree by amicetin and tetracycline. Formation of an acLeu-pentanucleotide-ribosomes complex was strongly stimulated by sparsomycin.


Developmental study of the structure of sea urchin embryo and sperm chromatin using micrococcal nuclease.

Sea urchin embryo chromatin is hydrolyzed by micrococcal nuclease into a series of oligomers which are multiples of a monomer (repeating unit) containing 220 +/- 22 nucleotide pairs of DNA which accumulates during the initial phase of the digestion. Although the size of the chromatin monomers remains the same throughout early development, from the morula through the pluteus stage of embryogenesis, the rate and extent of solubilization of chromatin DNA by micrococcal nuclease decrease as development proceeds. Sea urchin spermchromatin is hydrolyzed by micrococcal nuclease into a series of oligomes which are multiples of a monomer containing 260 +/- 26 nucleotide pairs of DNA which accumulates during the initial phase of the digestion. Analysis of the sizes of oligomers which result form micrococcal nuclease digestion of mouse liver, sea urchin embryo, and sea urchin sperm chromatin in situ, suggests that the oligomers are nearly exact multiples of the respective monomers. These results are discussed in relation to those studies which have shown that the histone complement of the sea urchin embryo and sperm changes during development.


Inhibition of DNA synthesis by cycloheximide and blasticidin-S is independent of their effect on protein synthesis.

The effects of cycloheximide and related glutarimide antibiotics on DNA synthesis in Achlya bisexualis Coker and A. Couch were compared to those of other protein synthesis inhibitors, puromycin, p-fluorophenylalanine and blasticidin-S. The inhibitors had no significant effects on intrahyphal pool sizes of dTTP, dCTP, ATP, UTP and CTP, nor on the specific activity of the dTTP pool labelled by [3H] thymidine. DNA was the sole acid-insoluble product of [3H]-thymidine incorporation. Cycloheximide, isocycloheximide, streptimidone and blasticidin-S inhibited DNA synthesis rapidly and completely and anhydrocycloheximide was less effective. Cycloheximide acetate, puromycin and p-fluorophenylalanine did not inhibit DNA synthesis. It is concluded that the effects of the several glutarimide antibiotics and of blasticidin-S on DNA synthesis were independent of their effects on protein synthesis.


The torsional potential for phosphate diesters. The effect of geometry optimization in CNDO and ab initio molecular orbital calculations.

CNDO and ab initio calculations on the dimethyl phosphate monoanion demonstrate that geometry optimization can dramatically alter the relative energies of the conformational isomers of the phosphate ester. Thus, with geometry optimization (particularly of the diester RO-P-OR bond angle) the energy differences between the gauche-gauche, gauche-trans, and trans-trans conformations is less than 1 kcal/mol rather than approximately 7 kcal/mol previously reported. A torsional energy map for the two ester torsional angles which includes ester bond angle optimization is presented. Ab initio (STO-3G level) and CNDO calculations confirm the strong coupling of the diester RO-P-OR bond angle to the torsional conformation.


DNA polymerase from mesophilic and thermophilic bacteria. III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus licheniformis and Bacillus stearothermophilus.

1. DNA polymerase from the mesophile Bacillus licheniformis and the thermophile Bacillus stearothermophilus has been used to study the replication of poly(dA-dT)-poly(dA-dT) and poly(dC)-poly(dG) templates at 37, 45, and 55 degrees C. 2. Incorporation of non-complementary deoxyribonucleoside triphosphates (misincorporation) occurred with both enzymes and both templates. Non-specific incorporation (de novo polynucleotide synthesis, random attachment to existing strands, and tritium exchange of nucleotides) accounted for, at most, a small fraction of the total observed misincorporation. The error rates at 37 degrees C for the complete system were as follows:: B. licheniformis: dATP, 1/61; dCTP, 1/830; dGTP, 1/360; dTTP, 1/65; B. stearothermophilus: dATP, 1/68; dCTP, 1/1430; dGTP, 1/440; dTTP, 1/67. For both organisms, the error rate for dCTP and dGTP was independent of incubation temperature; the error rate for dATP and dTTP was 5-50-fold greater than that for dCTP or dGTP and increased significantly from 37 to 55 degrees C. 3. The ratio of dATP to dTTP incorporation with the poly(dA-dT)-poly-(dA-dT) template was independent of temperature and close to unity. The ratio of dCTP to dGTP incorporation with the poly(dC)-poly(dG) template decreased from approx. 0.2 to 0.05 for the mesophile and from approx. 0.06 to 0.03 for the thermophile as the temperature increased from 37 to 55 degrees C.


Characterization of a DNA polymerase associated with an endogenous DNA-synthesizing complex isolated from human lymphoid cells.

We have shown that a membrane fraction prepared from isolated human lymphoid nuclei contains endogenous DNA-synthesizing activity which is sensitive to RNAase. We have isolated a DNA polymerase from this fraction and partially purified it to what we estimate as about 10 000-fold. Its chromatographic behavior, template specificity, sedimentation constant, pH optimum, and sensitivity to N-ethylmaleimide suggest that the activity resembles but is not identical to DNA polymerase gamma (formerly called R-DNA polymerase). The membrane fraction also contains a minor activity which is due to polymerase beta, the low molecular weight (3.5 S) nuclear enzyme.


Fluorescence of actinomycin D and its DNA complex.

In order to show that naturally occurring actinomycin D can be used as a fluorescent cytochemical probe, the fluorescence spectra of 50 micronM solution of actinomycin D in several solvents and in its DNA complex are reported. The excitation spectra of both bands observed are compared to the absorption spectrum, and the fluorescence quantum yields is estimated to be 0.65-10(-4), taking rhodamine B as a reference. Strong solvent-induced spectrum modifications are evidenced, interpreted as a solvent shell reorientation about the molecule chromophore in the excited state. The changes in the fluorescence spectrum of actinomycin due to an interaction with DNA (marked blue shift and decreased quantum yield) cannot be interpreted as a solvent effect; they express the properties of the DNA-chromophore eta-complex involving an orbital overlapping of actinomycin and guanine in DNA.


Isolation of A-T-rich fragments from calf thymus DNA using the phenol method.

The action of phenol on the products of partial digestion of calf thymus DNA by K2 DNAase causes a loss of 3--5% of the material passing into the phenol phase. This part of DNA can be regained in the aqueous phase by lowering both the temperature and the ionic strength. Among oligonucleotides up to 7 monomers in length, those which are soluble in phenol do not contain guanine residues. Phenol-soluble DNA fragments of the molecular weight of an order of 2000--50,000 appeared to be composed mainly of adenosine phosphate. They also contain some thymine and only traces of guanine and cytosine. Some longer A-T-rich fragments, even up to 5-10(6) daltons, were repeatedly found in the phenol phase, but their base composition has not been determined yet. The method presented here was found very convenient for the isolation of relatively large A-T-rich DNA fragments. The property of DNA or its fragments to dissolve in phenol seems to be dependent on an adequate primary structure, probably similar to that of poly (dA-dT).


Characteristics of the insulin stimulation of DNA, RNA and protein metabolism in cultured human mammary carcinoma cells.

In a mammary gland cell line (MCF-7) of human origin, insulin stimulated the rates of RNA, DNA and protein biosynthesis. These effects were observed with concentrations of insulin ranging from 10(-6) M to 10(-10) M. Enhanced rates of [3H]leucine incorporation into protein and [3H]uridine incorporation into RNA were observed within 1 h after exposing the cells to insulin. In contrast, a stimulatory effect of insulin on the rate of [3H]thymidine incorporation into DNA was only detectable following a 12--16 h incubation with insulin. Insulin also enhanced the rate of uptake of [3H]leucine, [3H]uridine, and [3H]thymidine into the MCF-7 cells. Finally, incubation with insulin increased the total amount of DNA, RNA and protein in these cells.


Age-related changes of accuracy and efficiency of protein synthesis machinery in rat.

The accuracy and the efficiency of the protein synthesis machinery has been measured by the variability of the [3H]leucine/[14C]phenylalanine ratio in a cell-free protein synthesis system, poly(U) directed, and with the same criteria by binding aminoacyl tRNA-ribosome complexes with poly(U). Using these two methods, we have observed the aging of rats to be characterized by an increase of 3H/14C ratios according to the "general error theory". The results obtained are discussed in relation to the redundancy and rectification of the genes involved in the protein synthesis machinery formation.


Haemoglobin J Guantanamo (alpha 2 beta 2 128 (H6) Ala replaced by Asp). A new fast unstable haemoglobin found in a Cuban family.

Haemoglobin J Guantanamo (alpha 2 beta 2 128 (H6) Ala replaced by Asp) was found during a screening from abnormal haemoglobins in three members of a Cuban family, of negro ancestry. The substitution in this variant is located at the alpha 1 beta 1 contact. This explains the slight instability and the mild haemolytic anaemia and morphological abnormalities found in the carriers of this variant. The instability of haemoglobin J Guantanamo indicates that the presence of Asp at the position beta-128 (H6) weakens the alpha 1 beta 1 contact.


A spectrophotometric study of the reaction of boro-hydride with trinitrophenyl derivatives of amino acids and proteins.

The absorption spectra of trinitrophenyl derivatives of poly(L-lysine) and L-asparaginase undergo irreversible changes in the presence of KBH4. The spectra of trinitrophenyl derivatives of N-acetyl-L-lysine and N-acetyl-L-cysteine are also affected by the addition of the reducing agent. A broad absorption band with a maximum at 426 nm appears in the presence of low concentrations of borohydride with a concomitant decrease in absorbance of the 346 nm band which is characteristic of 1-substituted 2,4,6-trinitrophenyl compounds. In the presence of higher concentrations of KBH4 the long wavelength band becomes less broad as the maximum is shifted to 410 nm and the 346 nm band completely disappears. Similar spectral changes were observed in the presence of Na2SO3 although these were reversible upon removal of the sulfite by dialysis. Based on the spectral similarities with sulfite and hydroxide adducts, we suggest that the 426 nm maximum represents a 1:1 adduct formed between the trinitrophenyl moiety and a hydride ion while the band at 410 nm is assigned to the 1:2 adduct.


Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin.

Using sequential pronase digestions, glycopeptide fractions were prepared from human alpha1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from alpha1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sailic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of --6400 deg-cm2-dmol-1 to --7200 deg-cm2-dmol-1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of --29 200 deg-cm2-dmol-1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex of glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetyl-glucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.


Hemoglobin Pontoise alpha63 Ala replaced by Asp(E12). A new fast moving variant.

The present report describes structural and functional studies of a new hemoglobin variant, Hb J Pontoise. This abnormal hemoglobin was discovered in a 36 year old Spanish man. The substitution of alpha63 (E12) Ala replaced by Asp is situated at an external site and appears to have no influence on the function of the hemoglobin. Nevertheless, it presents some instability in vitro, as demonstrated by the isopropanol test. This instability could partially explain the low percentage of the abnormal hemoglobin in the hemolysate (12%), since its rate of synthesis was normal.


Staining of proteins after isoelectric focusing in gels by new procedures.

Four procedures are described for staining of protein bands after isoelectric focusing in polyacrylamide gels. In one procedure, Coomassie Brilliant Blue R 250 is used whereas in the others the G 250 form is used in an aqueous solution of perchloric acid. By using isoelectric focusing and the new staining procedures it is possible to detect less than 0.2 microng of many proteins. One of the procedures is especially rapid, allowing detection of proteins without destaining. Procedures for preservation of the protein patterns and determination of proteins by scanning in a densitometer are also described.


Purification and characterization of a basic protein from the stratum corneum of mammalian epidermis.

A basic protein has been isolated and purified from the stratum corneum of newborn rat epidermis. This protein is referred to as stratum corneum basic protein. It was purified by ion-exchange chromatography on DE-52 and CM-52 cellulose. The protein has a molecular weight of 50 000 on sodium dodecyl sulfate-polyacrylamide gels. It is composed of one polypeptide chain and contains no detectable carbohydrate. The protein has an isoelectric point in the range of pH 9-10, but decomposes during isoelectric focusing giving rise to a polypeptide of less than 10 000 daltons. Amino acid analysis reveals high quantities of glutamic acid, glycine, serine, arginine and relatively high levels of histidine, with these five amino acids composing 74% of the total residues. The amino acid analysis is very similar to histidine-containing keratohyalin proteins isolated from the granular layer of epidermis by several investigators. The stratum corneum basic protein differs from fibrous proteins isolated from the same cell layer with respect to net charge, amino acid composition, and molecular weight. The protein does not react with antibody to the fibrous protein. The basic protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein.


Labeling of a major fibroblast surface protein (fibronectin) catalyzed by blood coagulation factor XIIa.

Incubation of cultured human fibroblasts with blood coagulation factor XIIIa (plasma transglutaminase, fibrinoligase) and the fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, resulted in fluorescent labeling of three cellular polypeptides. The molecular weights of the labeled polypeptides, estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction, were: greater than 1.2-10(6), 2.2-10(5), and 1.3-10(5). The labeled 2.2-10(5) dalton polypeptide was susceptible to mild trypsinization and not present in cultures of SV-40 transformed fibroblasts, indicating that it is the subunit of cell-surface fibronectin and identical with the external transformation-sensitive polypeptide of similar molecular weight described by others. Upon coelectrophoresis, the labeled 2.2-10(5) dalton polypeptide migrated slightly behind the subunit of plasma fibronectin (cold-insoluble globulin), indicating that the immunologically cross-reactive forms of fibronectin in human plasma and cultured human fibroblasts differ slightly in molecular weight. The identities of the labeled greater than 1.2-10(6) and 1.3-10(5) dalton polypeptides are not known. The XIIa-reactive glutamine residues of fibroblast cell-surface proteins are potential sites for intermolecular cross-linking (by xi-(gamma-glutamyl)lysyl linkages) to other proteins of connective tissue.


Copper-thionein from fetal bovine liver.

It was of interest to examine whether or not a low molecular weight copper-rich metal-thionein was present in biological species which received no metal pretreatment at all. From bovine fetal liver an 8 Cu 2 Zn-thionein having a molecular weight of 11 500 was successfully isolated. 16% of the total copper present in the whole liver were recovered in this protein. During the isolation process anaerobic conditions had to be maintained to avoid uncontrolled oxidation leading to polymeric species and the loss of most of the copper. The similarity of both the present copper-thionein and the polymeric neonatal type mitochondrocuprein was shown. A comparison of different copper-thioneins containing variable amounts of copper was possible when xiCu from 280 nm to longer wavelength was determined. With respect to the ultraviolet properties there were no detectable differences between copper-thioneins prepared either in vivo or in vitro and the fetal copper-thionein. Furthermore, the positions of the Cotton effects as deduced from circular dichroism measurements were rather similar although the magnitude of the observed Cotton extrema was less pronounced and sometimes the signs were reversed. X-ray photoelectron spectrometric studies revealed a Cu(2p3/2) binding energy value of 932.9 eV. Unlike the S(2p1/2,3/2) value near 162 eV using Cu-thioneins from chicken liver or yeast the higher S(20p1/2,3/2) binding energy of 163.0 eV employing fetal Cu-thionein was attributed to partial oxidation of the protein moiety and/or a particular chemical environment. The second S(2p1/2,3/2) peak was assigned to the copper catalyzed oxidation of sulphur via OH to yield RSO-3. In the X-ray photoelectron spectrum of the apoprotein one homogeneous S(2p1/2,3/2) band at 163.7 eV was seen attributable to RSSR.


The myoglobin of the killer whale (Orcinus orca).

Whereas the myoglobin of the dolphin surprisingly resembles that of the sperm whale more than that of the porpoise, the myoglobin of a killer whale does not show this feature. It differs from the myoglobins of the dolphin in 6, of the porpoise in 8, and of the sperm whale in 14 positions. The residues in which the cetaceans differ are localised in one particular area of the molecule.


Reactions of polyfunctional amino acids with N,N'-carbonyldiimidazole in aqueous solution--oligopeptide formation.

1. Serine reacts with N,N'-carbonyldiimidazole in aqueous solution at 0 degrees C to yield Nalpha-[imidazolyl-(1)-carbonyl]-L-serine. This compound slowly transforms to L-2-oxo-oxazolidine-5-carboxylic acid. We found that L-2-oxo-oxazolidine-5-carboxylic acid was stable to hydrolysis under a variety of conditions and did not oligomerize to yield peptides. Threonine was found to react in an analogous manner with N,N'-carbonyldiimidazole, yielding Nalpha-[imidazolyl-(1)-carbonyl]-L-threonine and L-(+)-trans-5-methyl-2-oxo-oxazolidine-5-carboxylic acid. 2. Histidine reacts with N,N'-carbonyldimidazole in aqueous solution at 0 degrees C to yield a variety of histidine-containing intermidiates. These slowly transform to give 7-carboxy-imidazole-[1,5cotetrahydropyrimidin-5-one in up to 90% yield. 3. The polymerization of 7-carboxy-imidazo-[1,5c]-tetrahydropyrimidin-5-one in imidazole buffer at 30 degrees gives excellent yields of oligohistidines.


Isolation and properties of prophospholipase A2 and phospholipase A2 from horse pancreas and horse pancreatic juice.

Two phospholipases A2 (EC 3.1.1.4) with different isoelectric points have been isolated from horse pancreas in high yield (880 mg/kg tissue). From pancreatic juice the more acidic species was isolated as the sole phospholipase A2. Upon tryptic activation the zymogens release a hepta- and pentapeptide, respectively from the N-terminal part of the protein giving rise to the formation of one single enzyme with a specific activity higher than that of pancreatic phospholipases A2 from other mammalian species. Horse phospholipase A2 differs from the porcine and bovine enzymes with respect to amino acid composition and kinetic properties. The sequence of the first 41 amino acid residues at the N-terminus has been determined by automatic Edman degradation.


Isolation of myocardial depressant factor from plasma of dogs in hemorrhagic shock.

Starting with 31 of plasma from dogs in hemorrhagic shock, we have purified the myocardial depressant factor and found that the activity is separate from salts and free amino acids. Moreover, the myocardial depressant factor is present in shock plasma in concentrations of about 1 nmol/ml of plasma. The depressant factor exists as multiple chromatographic forms. The best characterized forms are the anionic forms. A preliminary amino acid composition of the anionic forms has been obtained. These findings should allow more rapid processing of plasma containing high myocardial depressant factor activity to separate the factor and to completely identify this small peptide of great physiologic interest.


Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A.

Tryptophan residues of diphtheria toxin fragment A have been modified by 2-hydroxy-5-nitrobenzyl bromide. Fragment A loses its ability to inactivate ADP-ribosylation of the elongation factor 2, as a function of the number of residues modified. Modification of one tryptophan residue provides a dramatic loss of enzymic activity suggesting the presence of one essential residue of this type in fragment A. Examination of fingerprint maps of chymotryptic peptides of fragment A after reaction with 2-hydroxy-5-nitrobenzyl bromide allows us to identify this tryptophan residue at position 153 of the amino acid sequence. ORD and CD experiments joined to immunochemical studies seem to exclude the possibility that the observed decrease in the enzymic activity may be attributed to a conformational change. Finally, results of nicotinamide-adenine dinucleotide-binding measurements suggest that tryptophan 153 would be concerned with the elongation factor 2 binding site or with the catalytic site itself.


N- and C-terminal amino acids of purified alpha-actinin.

Highly purified bovine cardiac alpha-actinin is obtained by successive chromatography on DEAE-cellulose and hydroxyapatite of a crude fraction obtained by salting out low ionic strength extracts of bovine cardiac muscle between 0 and 30% ammonium sulfate saturation. Hydroxyapatite chromatography removes a 43 000-dalton polypeptide chain that is difficult to remove by successive DEAE-cellulose columns. Removal of all 43 000-dalton material by hydroxyapatite chromatography is accompanied by disappearance of a very small 9 to 10 S boundary in analytical ultracentrifuge diagrams of DEAE-cellulose-purified 6.2S alpha-actinin. Approximately 95% of the protein in DEAE-cellulose and hydroxyapatite-purified alpha-actinin is the 100 000-dalton alpha-actinin polypeptide as estimated by SDS-polyacrylamide gel electrophoresis. Purified bovine cardiac, porcine skeletal, chicken gizzard, and chicken breast alpha-actinins all contain leucine as the C-terminal amino acid of both polypeptide chains in the alpha-actinin molecule. Bovine cardiac and porcine skeletal alpha-actinins contain arginine as the amino acid penultimate to C-terminal leucine. None of the four different alpha-actinins studied had a N-terminal amino group available for reaction with dansyl chloride, but all four alpha-actinins contained 1.6 to 1.8 acetate residues per molecule (200 000 daltons) of alpha-actinin. It seems likely that the N-terminal amino groups of both polypeptide chains in these four alpha-actinins are acetylated. A peptide having the composition N-Ac-Asp2-Glu4 was isolated from a proteolytic digest of bovine cardiac alpha-actinin. alpha-Actinin seems to be a conserved protein molecule found in many different motile systems.


Evaluation of secondary structure predictions in proteins.

Data of 33 proteins are used to compare four methods which predict secondary structure from the amino acid sequence. The prediction of alpha-helices according to the histogram method of Argos et al. (Argos, P., Schwarz, J. and Schwarz, J. (1976) Biochim. Biophys. Acta 439, 261-273) is on the average as reliable as the statistical method of Nagano (Nagano, K. (1973) J. Mol. Biol. 75, 401-420). Both methods predict helices more accurately than the stereo-chemical method of Lim (Lim, V.I. (1974) J. Mol. Biol. 88, 873-894). The method of Nagano yields the best prediction of beta-structure, while the beta-structure predictions of Lim and Argos et al. are not significantly different. The results of the alpha-helix and beta-structure predictions according to the statistical mechanical method of Tanaka and Scheraga (Tanaka, S. and Scheraga, H.A. (1976) Macromolecules 9, 168-182) are inferior to those obtained by the other three methods. For the prediction of turns, no significant difference between the methods of Nagano (Nagano, K. (1974) J. Mol. Biol. 84, 337-372) and Argos et al. was found.


The temperature-dependent dissociation of spectrin.

Spectrin, isolated from both human and bovine erythrocyte membranes, was found to exist predominantly as a tetramer of 960 000 daltons when prepared at 4 degrees C, but as a dimer of 480 000 daltons when prepared at 37 degrees C. The purified tetramer from both species could be dissociated to dimers by low ionic strength incubation at 37 degrees C.


The importance of protein structure and conformation in the preparation of phospholipase-free cardiotoxin from snake venom.

Hydrophobic interactions of cobra venom phospholipase A2 (Mr 13 400) in saline with Sephadex gels and its stability towards denaturation in 6 M guanidine-hydrochloride precluded the use of these solvents to remove traces (approx. 0.2%, w/w) of phospholipase A2 from cardiotoxin (Mr 6800) by gel chromatography. Phospholipase-free (less than 0.001%, w/w) cardiotoxin could, however, be obtained by gel chromatography in 8 M urea or 80 mM phenylalanine. Stokes radius and circular dichroism measurements showed that the hydrophobic retardation of phospholipase A2 was abolished but that the hydrodynamic size and conformation of neither protein was affected, thereby facilitating separation.


Primary structures of cardiotoxin analogues II and IV from the venom of Naja jaja atra.

Cardiotoxin analogues II and IV were isolated from the venom of Naja naja atra by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The venom contains at least four cardiotoxin analogues that account for about 54% of the weight of the lyophilized crude venom. These four cardiotoxin analogues, named cardiotoxin analogues I, II, III, and IV, show strong cytotoxicity to Yoshida sarcoma cells but the lethal toxicity is one-order less. These toxins contain 60 amino acid residues in a single peptide chain. Cardiotoxin analogue IV differs from cardiotoxin analogue II only by the presence of arginine in place of a leucine residue at position 1. A comparison of the amino acid sequences of these toxins with that of cobrotoxin, a neurotoxin containing 62 amino acid residues obtained from the same snake venom, shows that about 20 amino acid residues, including 8 half cystine residues, are identical, assuming 3 residues deletion and 2 residues insertion in the cardiotoxin molecule.


Structural features of rat cardiac ferritins.

Ferritin extracted from rat heart containes two species separable by gel electrophoresis. These were purified and examined for structural characteristics. As in gel electrophoresis, cardiac ferritin preparations yielded only two bands on isoelectric focusing in gels, with pI values of 4.6 and 4.8. After separation by preparative electrophoresis, the two species were found to have a different amino acid composition from each another and from liver ferritin. Similarly, peptide maps showed several components not found in liver ferritin. On dissociation and electrophoresis with sodium dodecyl sulfate, heart ferritins were found to contain subunits of the same sizes as in other rat ferritins but also some larger components. Since cardiac ferritins have apparent molecular weights greater than those of other ferritins, it is concluded they probably contain more subunits, and possibly some of larger size not present in ferritins of other tissues.


Isolation and characterization of rat alpha-lactalbumin: a glycoprotein.

alpha-Lactalbumin was purified to homogeneity from rat milk. Rat alpha-lactalbumin, in contrast to other alpha-lactalbumins, is a glycoprotein and exhibits an abnormally high molecular weight when obtained by gel filtration or electrophoresis in sodium dodecyl sulfate. The molecular weight by sedimentation equilibrium is 15 400 +/- 5% and of the reduced and alkylated protein is 16 000 when determined by thin-layer chromatography in 6 M guanidine hydrochloride. At least, three major charge forms, all containing carbohydrate and active in the lactose synthetase reaction were demonstrated. The amino acid composition reveals a high proline content which is reflected in a low alpha-helical content.


Purification and some physicochemical properties of bovine kappa-casein.

1. A description is given of the fractionation of kappa-casein on DEAE-cellulose using a pH gradient. With this method an improved separation of the kappa-casein components with a higher negative charge is obtained. 2. It is shown that at least one of the kappa-casein fractions has a second phosphate ester group. The heterogeneity of kappa-casein therefore is not exclusively caused by a varying N-acetylneuraminic acid content. 3. Ultracentrifuge experiments and exclusion gel chromatography show that the purified kappa-casein fraction having the lowest electrophoretic mobility exhibits a monomer-polymer association equilibrium. The free energy of association per mol monomer in 0.2 M NaCl is approximately --36 kJ-mol-1.


Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver Golgi apparatus.

Neuraminidase and galactosyltransferase were investigated in total Golgi appartus and in the three fractions of increasing densities (GF1, GF2, and GF3) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45-72). About 50% decreases in neuraminidase content (units/g liver) and specific activity (units/mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (introperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123-125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed in Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.


Enzyme activities in the sheep placenta during the last three months of pregnancy.

In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and keton body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy. The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do not occur in the sheep placenta. It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.


Analysis of the carbon-13 and proton NMR spectra of bovine chromaffin granules.

Natural abundance carbon-13 and proton NMR spectra of bovine chromaffin granules have been obtained and analyzed using computer simulation techniques. High resolution spectra show the presence of a fluid aqueous phase containing epinephrine, ATP and a random coil protein. The protein spectrum contains unusually intense resonances due to glutamic acid and proline and has been simulated satisfactorily using the known amino acid composition of chromogranin A. The lipid phase of chromaffin granules gives rise to intense, but very broad, resonances in the carbon-13 spectrum. Protons in the lipid phase are also observable as a very rapid component of the proton-free induction decay (T2 approximately equal to 15 microns). Linewidths of the carbon-13 spectra have been used to set upper limits on rotational correlation times and on the motional anisotropy in the aqueous phase. These limits show that the aqueous phase is a simple solution (not a gel) that is isotropic over regions much larger than solute dimensions. No gel transition is observed between -3 and 25 degrees C. The carbon-13 spectra are definitely inconsistent with a lipoprotein matrix model and chromaffin granules previously proposed by Helle and Serck-Hanssen ((1975) Mol. Cell, Biochem. 6, 127-146). Relative carbon-13 intensities of ATP and epinephrine are not consistent with the known 1 : 4 mol ratio of these components. This fact suggests that epinephrine and ATP are not directly complexed in intact chromaffin granules.


Comparative studies of water sorption of hyaline cartilage.

Vapor phase, water sorption isotherms were obtained for specimens of bovine, sturgeon and shark cartilage and for membranes composed of collagen and various proportions of cartilage proteoglycan. The data were interpreted in the light of an elementary model for swelling of gels which regards equilibrium swelling a resultant of a balance between contractile forces of an elastic matrix and expansive forces, principally osmotic in nature. Swelling ratios for bovine and sturgeon cartilage compared at the same water vapor pressure are nearly identical, whereas the swelling ratios for shark cartilage are elevated. These high values are due principally to a higher ratio of glycosaminoglycan to collagen but also reflect a higher salt and urea content and possibly also a different type of collagen fibril network.


Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells.

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using 125I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (Ka) of about 5 - 10(8)--10 - 10(8)M-1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding for IgG1 (Ka about 45 - 10(8)M-1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7 : 1 ratio of these immunglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.


Leakage of sucrose from phosphatidylcholine liposomes induced by interaction with serum albumin.

Liposomes composed of rat-liver phosphatidylcholine rapidly lose entrapped sucrose when incubated in presence of blood or of solutions of bovine serum albumin. The phenomenon can not be ascribed to phospholipase A activity, since no such activity towards phosphatidylcholine substrates could be detected in various albumin preparations. Upon gel filtration on Sepharose 4B or Sephadex G-100 of incubated mixtures of radioactive liposomes and albumin, association of phosphatidylcholine with the albumin could be demonstrated. No measurable quantities of protein were found associated with liposomes. The albumin-associated phosphatidylcholine is hydrolyzed by pancreatic phospholipase A more slowly than free liposomal phosphatidylcholine, indicating a non-lamellar orientation of the associated phospholipid. The binding of phosphatidylcholine to albumin proceeds at a slow rate: increase of the amount of phosphatidylcholine bound continues over a period of several hours reaching a maximum at approx. 1 mol of phosphatidylcholine per mol of albumin. The process is reversible as indicated by transfer of albumin-associated radioactive phosphatidylcholine to unlabeled liposomes. The association between albumin and phosphatidylcholine is believed to be of the same type as described recently by Jonas (Jonas, A. (1976) Biochim. Biophy. Acta 427, 325-336). The consequences of these observations are discussed with respect to the use of liposomes as carriers to introduce substances into cells.


The influence of dietary selenium and vitamin E on glutathione peroxidase and glutathione in the rat.

The effect of dietary selenium (Se) and vitamin E supplementation on tissue reduced glutathione (GSH) and glutathione peroxidase activity has been studied in the rat. Increasing Se intake by 0.4 ppm gave significantly higher enzyme levels in all tissues studied, an effect not influenced by vitamin E intake. Further increasing Se to 4 ppm gave higher enzyme levels in red blood cells only, while in liver there was a significant decrease in enzyme activity probably reflecting Se hepatotoxicity. In the absence of Se supplements increasing dietary vitamin E to 100 mg/kg diet significantly increased enzyme activity but this effect was modified by simultaneous Se supplementation. Se intake had no effect on GSH levels. Rats on a high vitamin E intake 500 mg/kg had a significantly higher tissue GSH level. Dietary Se had a sparing effect on vitamin E, rats supplemented with Se having significantly raised plasma vitamin E levels. These results confirm the role of selenium in glutathione peroxidase and also show that vitamin E influences the activity of the enzyme.


Effect of methionine on the metabolism of formate and histidine by rats fed folate/vitamin B-12-methionine-deficient diet.

The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [2-14C]histidine and [14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.


Identification of glutamate chain lengths of endogenous folylpoly-gamma-glutamates in rat tissues.

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described. Natural pteroylpoly-gamma-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-gamma-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol:ammonia. Aminobenzoylpolyglutamates were separated by DEAD-cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines. The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.


Uptake and metabolism of [3H]folate by normal and by vitamin B-12- and methionine-deficient rats.

The uptake of an injected dose of [3H]folic acid and its metabolism to pteroylpoly-gamma-glutamate forms by the livers and kidneys of vitamin B-12- and methionine-deficient and -supplemented rats were investigated. The initial hepatic uptake of the labeled folate dose was the same in deficient and supplemented animals, demonstrating no involvement of vitamin B-12 or methionine in folate transport. At longer time periods, a decreased hepatic net uptake of labeled folate was observed in the deficient animals compared to supplemented animals, and this was directly correlated with the decreased ability of the deficient animals to synthesize pteroylpolyglutamates. The absolute rate of loss of labeled pteroylmonoglutamate from liver was the same in deficient and supplemented animals. These data are best explained by a modification of the 'methyl trap' hypothesis for the interrelationship of vitamin B-12 and folate metabolism. Vitamin B-12 deficiency can lead to lowered levels of 5-methyltetrahydrofolate:homocysteine methyltransferase, creating a functional folate deficiency by 'trapping' an increased proportion of folate as the methyl derivative. In addition, as methyltetrahydrofolate is a poor substrate for folylpoly-gamma-glutamate synthetase, there is a decreased synthesis of pteroylpolyglutamates, the forms of the vitamin that are preferentially retained by tissues. This results in decreased tissue folate levels under conditions of vitamin B-12 deficiency. Vitamin B-12 and methionine deficiency had no significant effect on the distribution of endogenous pteroylpolyglutamates in rat liver and kidney, although total endogenous folate in rat liver was reduced by about 60%. The distribution of labeled pteroylpolyglutamates in rat liver and kidney 48 h after the tracer dose of [3H]folate closely resembled the endogenous distribution in these tissues.


Molecular mobilities of soluble components in the aqueous phase of chromaffin granules.

NMR relaxation times have been used to characterize molecular motion and intermolecular complexes in the aqueous phase of bovine chromaffin granules. Partially relaxed 13C and proton spectra have been obtained at 3 and 25 degrees C. T1 measurements of five protonated carbons on epinephrine (C2, C5, C6, CHOH and NCH3) give a correlation time of 0.15 (10(-9)) s at 25 degrees C for the catechol ring and methine carbon, while the effective correlation time for the NCH3 group is somewhat shorter due to its internal degree of rotational freedom. Resonances of protonated carbons on the soluble protein chromogranin give very similar correlation times: 0.20 (10(-9)) s for the peptide alpha-carbon and 0.2 (10(-9)) s for the methylene sidechain carbons of glutamic acid. The correlation time (tauR) of ATP was not measured directly using 13C T1 data due to the weakness of its spectrum, but its reorientation appears to be substantially slower than that of epinephrine or chromogranin. This conclusion is based on three observations: (1) the qualitative temperature dependence of T1 for H2 and H8 on the adenine ring places tauR for ATP to the right of the T1 minimum, or tauR greater than or equal to 1.0 (10(-9)) s; (2) 13C-resonances of ATP have anomalously low amplitudes compared with epinephrine resonances, a fact that is readily explained only if ATP undergoes substantially slower reorientation; and (3) a comparison of the T1 data of H8 in chromaffin granules and in a dilute aqueous solution, where tauR for ATP can be measured directly indicates that tauR approximately 1.0 (10(-9)) s at 25 degrees C in the granules. The relaxation data are consistent with the concept of a storage complex based on electrostatic interactions between a polyion (chromogranin) and its counterions (ATP and epinephrine), in which ATP cross-links cationic sidechains of the protein.


Sex difference in distribution and iron responsiveness of the two ferritins of rat cardiac and skeletal muscle.

The presence of two electrophoretically and structurally distinguishable forms of ferritin ("fast" and "slow") in cardiac and skeletal muscle (diaphragm) of the rat was confirmed. Although the total amount of cardiac ferritin showed no difference in concentration in male and female rats, the distribution between the fast and slow species was markedly different in the two sexes, the fast form predominating in the cardiac muscle and diaphragm of the female. In agreement with this, the rates of synthesis and of degradation of the fast species were greater in the female, while the opposite obtained for the male. Iron administration stimulated synthesis of each ferritin species in the cardiac muscle and diaphragm of both sexes. Induction of cardiac connective tissue hypertrophy with isoproterenol inverted the ratio of slow to fast ferritin in female rats, while iron administration along with isoproterenol restored this to normal. It is concluded that the metabolism of ferritin in cardiac and skeletal muscle is sensitive both to sexual status and to iron administration.


The aggregation of basic polypeptide residues bound to heparin.

The molecular basis for heparin interactions with proteins has been explored with L-lysine copolymer : heparin complexes, measuring the conformational change and charge neutralization which accompany the complexation, using optical methods. Previous studies had shown that the basic homopolypeptides (poly-L-lysine, poly-L-arginine) assume alpha-helical conformation upon interaction with numerous glycosaminoglycans (including heparin). Thus, the unique specificity for heparin in the anticoagulation system (which involves two or more lysine residues on the antithrombin molecule) is not paralleled by the findings with the basic homopolymers. Results with mixed polypeptides, poly(lysine : tyrosine, 1 : 1) and poly(lysine : phenylalanine, 1.4 : 1), show that these protein models assume different conformational forms upon complexation with heparin, the former shows a poly-L-lysine-like beta-structure circular dichroism spectrum and the latter an alpha-helical structure. The change in circular dichroism spectra increases with the addition of heparin until the ratio of positive to negative charge is about one. Dye-binding studies of the two copolymer systems reveal that the charged groups of both reactants are largely blocked in the polypeptide complexes at a calculated charge ratio equal to one. The data indicate that heparin interaction with the cationic polypeptides causes them to assume either the alpha-helical or beta-structure depending upon the nature of the neighboring uncharged amino acid and its proclivity for alpha-helix or beta-structure.


Possible evidence for the existence of a growth hormone fragment in porcine pituitary.

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.


Correlation of relaxation time with water content in muscle and brain tissues.

Proton T1 of water in muscle and brain tissues was studied as a function of water content at 25 degrees C and 5, 30, and 100 MHz. Muscle samples were bathed in modified Krebs solutions that have the same ionic strength but different osmolarity in order to change the water content. At all three frequencies their 1/T1 extrapolated to the same value of 0.6 S-1 for zero dry weight, indicating that T1 for the majority of water molecules in normal muscle and brain is frequency-independent. Dehydrated muscle, homogenized brain tissue suspended in modified Krebs solution and dehydrated brain tissue provide samples with wide ranges of water content. Their T1 values at all three frequencies can be well accounted for by a simple equation derived from the two-state model.


[Analysis of electrophysiologic characteristics of the frog atrial trabecular membrane by ionic currents at a fixed potential].

The membrane of frog auricle trabecula was studied by the method of potential fixation. On the basis of the records of ionic currents zyro-isoclines of approximative differential equations of the 2nd order, describing the membrane studied, were plotted. The mathematical model of this membrane was analysed with the help of qualitative methods of the oscillation theory. The analysis has shown that the following phenomena which have not been observed experimentally should take place in the membrane of frog auricle trabecula: 1. absence of the minimum gradient and accomodation growth of thresholds; 2. absence of anode-disconnecting excitation; 3. coincidence of the current of repeated responses with that of the rheobase; 4. uneven depolarization.


[Intercellular coupling in the sinus node of the rabbit heart].

Passive electrical properties of SA node region of rabbit's heart were studied. Electrotonic anisotropism was characteristic of the entire SA node; anisotropism decreased moving from crista terminalis to interatrial septum. The input resistance, RBX, was the largest in the region of true pacemakers and decreased moving to the perinodal region. Analysis of the results yielded specific resistance values for contacts and electrogenic membrane of 3 Ohms-cm3 and 10(4) Ohms-cm2 respectively.


[Instability and propagation of waves of excitation in a model of a catalytic reaction. III. Non-trivial regimes of propagation of excitation].

The regime of excitation propagation in a uniform model of an active medium is studied within the parameters where the initial concentrated system is a trigger one. In a distributed system propagation trigger waves as well as impulses can propagate. A means for initiating such impulses is suggested, an unstationary regime of their imitation is investigated by calculations. Piece-linear solutions obtained analytically are shown to be a good approximation for stationary regimes.


[Analysis of spatial frequency characteristics of complex receptive fields].

The difference fB2-fB1=deltaf and ratio fB2/fB1=R of the bandwidth boundary frequencies fB1, fB2 of spatial frequency characteristic of complex receptive field depends on the size of RF and spatial structure of excitatory and inhibitory zones. deltaf and r are independent of the weighting functions of excitatory and inhibitory zones. The theoretical value of r is similar to data which was obtained in psychophysical and physiologycal experiments. The dependence of contrast sensitivity on spatial frequency can be explained by dependence of the envelope of weighting functions of exitatory (inhibitory) zones on the spatial frequency.


[Model studies of lateral inhibition as a mechanism of detecting motion. I. Analytic consideration of direct inhibition].

Physiological studies show that lateral inhibition is involved in movement detection and suggest that an essential role in this process is played by such factors as an initial delay of inhibition, final velocity of propagation and its gradual reduction as the inhibition mediator is disintegrated--"memory" of inhibition. A mathematical model of dynamic lateral inhibition is proposed to check up this suggestion and to determine the role of each of these factors. An analytical investigation of the direct inhibition model confirms the participation of these factors in the separation of the reaction to movement. It is found that the delay of inhibition and the final velocity of its propagation are the factors, which favour movement separation and do not impose restrictions on special characteristics of inhibition providing for the fulfilment of the known functions of stationary lateral inhibition. Restrictions appear in the case when movement separation is determined by the delay and "memory" of inhibition.


[Model of the system for stabilizing the frequency of the echosignal of the Rhinolophus locator].

Physical analysis of anatomic, physiological and behavioural data pertaining to the work of Rhinolophidae echolocator is carried out, results of spectral analysis of its probing impulse are analysed. On this basis the working mechanism of sound emitting apparatus is proposed. A phenomenological model of the frequency stabilization system of the signal reflected from a located object is constructed. This stabilization is carried out by the compensation of Doppler shift of the echo frequency by a change of the generated impulse frequency. Due to the application of a differential method the model provides for potential accuracy of tuning, and its work does not depend on the value of a reflected signal and its sufficiently small changes which is in agreement with the experiments.


[Energy relationships in the human cutaneous system under conditions of single, residual and reverse masking].

Studies were carried out by electron-mechanic adequatometer permitting to obtain two shifted in time air stimuli. According to the curves of pressure duration thershold energies were determined theoretically for the stimuli of various durations; energy-duration curves were plotted. The adequatometric analysis of the curves made it possible to determine: 1. qualitative and quantitative similarity of energetic regularities of simultaneous residual and reverse masking; 2. the masking interval value during the residual masking; 3 critical time of discovering the threshold stimulus during the reverse masking.


[Structure of the spectrum of dolphin communication signals].

Dynamics of changes in the thin structure of the spectrum of dolphin communication signals is studied using Furie algorythm of fast transformation. Computer analysis of whistle signals allows to obtain quantitative evaluations of spectral characteristics and shows that coding of information in dolphin communication signals may be carried out by controling of signal harmony power and multiplicative modulation of frequencies of the basic tone and harmony.


[Component breakdown of the absorption and fluorescence spectra of green bacteria].

The total number, band widths, absorption and fluorescense peak positions of bacteriochlorophyll forms in green bacteria were identifyed using computer curve analysis, Stepanov's relation and low temperature derivative spectroscopy. The identified pigment forms are the following (the lower index--absorption peak position, the upper--fluorescence peak position) (see article). The energy of the O-O transitions in the row of native forms of bacteriochlorophyll enumerated above changes for a constant equal to 80 cm-1. It is believed that the bacteriochlorophyll aggregate in the reaction center complex of green bacteria consists of 7 molecules.


[Spatial-frequency characteristics of the receptive fields of cat visual cortex with different rates of stimulus movement].

Velocity ranges exist for complex and simple receptive fields of cat's visual cortex. In these ranges there is a narrow band frequency tuning. In these velocity ranges the location of the maximum on frequency characteristics is constant and does not depend on the velocity of stimulating lattice movement. An increase of the movement velocity brings about a reduction of the reaction value of the receptive fields and a disappearance of the narrow band tuning expressed in the optimal velocity range.


[Thermodynamic properties of biopolymers in spiral and coiled states in the 4--400 degree K temperature interval].

Possibilities of the adiabatic calorimetry methods in the studies of the biopolymer conformational properties in the wide temperature range including helium temperatures are considered. The low temperature peculiarities of the heat capacity difference for the biopolymers being in helical and coiled states are found. It was shown, that in the presence of biomacromolecules the water in the ice-water phase transition region suffers gradual over the temperature phase transition "order-disorder". It was found, that thermodynamic properties of the linear fibrillar structures over the wide temperature range (including helium temperatures) are essentially defined by the state of molecule solvent built-in the biopolymer helical structure. New data on the interaction mechanisms of dissolved salt ions with the biomacromolecules in the native and denaturated states have been obtained. It is shown that the existence of different fractions of bound water with biopolymer is a fact. A conclusion was made that it is necessary to take into account the data on the thermodynamic properties of biomacromolecules and the water bound to them at low temperatures in order to described completely the conformational properties of biomacromolecules.


[Activity of lateral reticular nucleus neurons during scratching].

The activity of neurones of the lateral reticular nucleus (LRN) was recorded in thalamic cats immobilized with Flaxedil during "fictitious scratching", that is during rhythmical activity of motoneurones typical of normal scratching. The LRN neurones exhibited a distinct modulation of their discharge related to the rhythmical activity of motoneurones in spite of the absence of the rhythmical afferent inflow. The pattern of modulation was identical both in normal and in "fictitious" scratching. Therefore, the spino-reticulo-cerebellar path transmits information about the activity of the spinal motor centres.


[Study by the proton magnetic resonance method of complex formation between nucleosides and compounds modeling amino acid residues of proteins in dimethyl sulfoxide].

Proton magnetic resonance was used to investigate in dimethyl-sulfoxide specificity of complex formation between nucleosides and a number of substances modelling of carboxyl, guanidine and amide side chain residues of aminoacids. It was observed downfield chemical shift of NH2 group of guanosine and disappearence. NH groups resonance for all nucleosides containing its in heterocycle in complex with carboxyl group. The next selectivity of interaction of nucleosides with carboxyl was observed: Ino greater than Guo greater than Urd ==Thd greater than Ado==Cyd. Obtained data indicates formation specific hydrogen bonds between carboxyl group and NH2 group of guanine and more strong hydrogen bonds with NH groups of all bases. In the case of guanidine group there are more weaker interaction and for amide group complex is not observed at all.


[Structure of histone fragments and their models. I. Structural features of N-terminal oligopeptides of histone H4].

Circular dichroism (CD) and infrared spectroscopy (IRS) studies of aqueous solutions of fourth N-terminal peptides of histone H4 with different chain length were carried out under various conditions. It was shown that all studied peptides had conformation of extended left-handed helix as well as poly-1-proline II at the acidic and neutral pH, in moderate ionic strength (0,15), in 80% ethanol, 0,2 M sodium dodecylsulphate, in 8 M urea and 5 M guanidinum hydrochloride. This conformation was changed by raising temperature, under transition to the range of basic pH and in the concentrated solutions of CaCl2 (5M).


[Effect of the nature of a monovalent counterion on the secondary structure of DNA is aqueous solutions].

Doxyribonucleates (DNMe) and deoxyribonucleic acid (DNA) were prepared by the ion-exchange method from Na+-salt from chicken erythrocyte DNA (DNS). (Here Me+ means Rb+, CS+, Na+, or NH4+). It was found that in aqueous solutions of DNMe in which the concentration of nucleic phosphorus was 8-10(5) mole/1 and the supporting electrolyte contained as an impurity only, the secondary structure of DNA was partly restored. This was confirmed by low values of the atomic extinction coefficient (epsilon260(p) = 6800 1/mole-cm and by high values of the hyperchromicity coefficients (1-28). The melting temperatures, as well as the width of the melting transition calculated from the melting curves, did not depend on the nature of Me+. Abnormally high melting temperatures for aqueous solutions of DNMe were noted. It was shown that the DNS - DNA transition was accompanied by spectrum changes that are typical of the denaturation process.


[Dimeric mechanism of the work of nigericin in bilayer lipid membranes].

The electric properties of the bilayer lethitin membranes have been studied in the presense of the antibiotic nigericin. When the antibiotic concentration is about 10(-6) M the condactivity of the BLM is increased upto 10(-7) ohm-1cm-2. The potassium ion concentration gradient gives rise to a transmembrane potential of the order of 40 mV per 10--fold concentration gradient with the side of the higher potassium concentration negative. The transmembrane potential produced by the hydrogen ion  gradient is a function of the potassium ion concentration which is equal on both sides of the membrane. In order to account for the experimental results obtained, a theoretical approach has been developed based on the assumption that charge is transported across the membrane by nigericin dimers.


Percutaneous transhepatic cholangiography using the "Chiba" needle--80 cases.

Using the Chiba needle for percutaneous transhepatic cholangiography, bile ducts have been visualized radiographically in 80% of 80 patients with cholestatic jaundice. The success rate was 94.4% in the 54 patients with dilated bile ducts due to an extrahepatic bilary obstruction, and 50% of the 20 patients with undilated ducts. Four patients developed ascending cholangitis with septicaemia on the same day as the procedure, and early surgery after visualization of obstructed bile ducts is recommended.  The technique was successful in three of five patients with sclerosing cholangitis. Percutaneous cholangiography using the Chiba needle is a convenient method of opacifying the biliary system in patients with severe large bile duct obstruction.


[Induction of fluorescence of a single chloroplast in an entire cell of the green alga Haematococcus pluvialis].

The fluorescense induction time-course of a single chloroplast in an intact cell of the green alga Haematococcus pluvialis has a line structure, which could not be observed in a suspension because of the superposition of the emissions of many cells. The time-course of the induction includes 12 well separated parts in the time range from several milliseconds to several minutes. Parameters of the fine structure depends on the physiological state of the cell, intensity and wave-length of the exciting light.


Bone thinning in frontal mucocele.

Sixteen cases of frontal mucocele are reviewed. Bone thinning leading to destruction of the sinus floor was shown to be a feature. It usually affects the superomedial margin of the orbital roof first, due to pressure on the area where the sinus wall is thinnest. Demonstrable thinning of either anterior or posterior sinus walls--a cardinal sign of frontal mucocele, is best shown by means of underpenetrated lateral radiographs. All other conventional radiographic signs of frontal mucocele, including lucency of the affected sinus, were found to be non-specific. The presence of an osteoma in a site likely to obstruct the frontonasal duct is a valuable adjunct to diagnosis.


[Kinetics of the reaction between hemoglobin and carbon monoxide. III. Existence of conformationally unequal Hb molecules during the process of CO binding and dissociation].

The experimental data concerning CO binding to Hb in linear kinetic experiments were compared with theoretical results obtained earlier for several models. The results indicate that during the processes of CO binding and dissociation there appears Hb and HbCO molecules in out-or equilibrium conformational states. The dissociation effective rate constants for these HbCO molecules, formed from Hb molecules in equilibrium conformational state are kdiss greater than or equal to 5.5 s-1. At the same time, the equilibrium HbCO complexes after conformational relaxation have much smaller values of kdiss near to the well known literature values, i.e. 0.015 s-1.


Origin of the right vertebral artery from the right common carotid artery: angiographic demonstration of three cases.

Three cases are presented in which the right vertebral artery originates from the right common cartoid artery. The literature is reviewed and the significance of this anomally is discussed. It is suggested that this anomally occurs more commonly than indicated by previous reports.


[Effect of the ionic composition of the environment on the intracellular potential of the alga Acetabularia].

Concentration of K+-, Na+-, Mg++-, Ca++-, Cl-ions defined in Acetabularia crenulata cell was the same as that of Acetabularia mediterranea, both were cultivated under the same conditions. At the variations of the ionic composition of the external medium it has been shown that the potential of Acetabularia (A.) cell depends on K+-, Cl-ions and alga A. can accumulate K+- Cl-ions and lose Na+, Mg++ ones when there is a deficiency of these ions in the medium. Na+-, Mg++-ions participation in the generation of the rest potential of Alga A. is insignificant. K+- ion is able to change the ionic permeability of the membrane. There is a possibility of the existance of essential H+-, -OH-, or HCO3- ions flux in A. cell.


Medical imaging by NMR.

Application of the new nuclear magnetic resonance (NMR) method of fast scan proton imaging is described. Cross sectional proton scans of a human finger in vivo are measured and reveal considerable anatomical detail, particularly of the soft tissue regions. Imaging of tumour tissue is also briefly discussed.


Immunological studies in patients with malignant melanoma of the uvea.

Antibodies directed against different components of ocular melanoma cells were identified by indirect immunofluorescence in the sera of 12 patients with histologically proven malignant melanoma of the uvea. Antibodies reacing with autologous surface membrane antigens were found only in the sera of the 5 patients with the smallest tumors. The sera from most of the patients with small, large, or extraocular tumor contained antibodies directed against autologous cytoplasmic components while about one-half of the tests for antibodies reacting with allogeneic cytoplasmic antigens were positive. The incidence of positive tests with cytoplasmic antigens, in 67 patients with clinically diagnosed benign uveal nevi and 56 other individuals not showing evidence of proliferative lesions of uveal melanocytes, was 25%.


The effects of ocusert pilocarpine on anterior chamber depth, visual acuity and intraocular pressure in man.

The duration of intraocular pressure reduction by Ocusert Pilocarpine was found to be 10 days for P20 and 12 days for P40. Ocusert Pilocarpine reduced visual acuity and anterior chamber depth less than the aqueous pilocarpine drops.


A clinical study of the autorefractor, an antomatic refracting device.

Four hundred eyes were examined with the Autorefractor, an automatic refracting machine, and the results compared with those found by standard techniques of clinical refraction with and without cycloplegia. Agreement was good for spherical power, cylindrical power and spherical equivalents, but less so for cylinder axes.


Results after vitrectomy in trauma.

We report the results of vitrectomy in 15 consecutive cases of ocular trauma operated on during one year. The cases are divided into three groups according to whether the main damage involved the anterior, posterior, or anterior segments. Of the 15 cases two thirds had a successful outcome. A penetrating injury and total retinal detachment with massive preretinal retraction were two features associated with poor results.


Pars plana vitrectomy.

Sixty-four consecutive pars plana vitrectomies were performed with the Douvas roto-extractor for anterior and posterior vitreous opacities. Eighty-four per cent of the anterior segment vitrectomy patients obtained improvement in visual acuity after surgery. Only 60% of the posterior segment patients obtained improvement in visual acuity, while only 45% of the eyes with active proliferative diabetic retinopathy had improved vision.


[Corneal response to 'off' in the intact rabbit eye].

The corneal electrical responses of the retina to light OFF are described in this report. The results reveal that high intensities of stimulation evoke an OFF response containing a positive wave. Since this wave was resistant to Sodium aspartate it is concluded that it arises from the receptor level. Negative wave are of double origin a) a negative potential evoked with low light intensities which is resistant to Sodium aspartate b) a second negative wave which is sensitive to the latter drug and thus arises from the internal layers of the retina. It is concluded that, because of the absence of a prominent b wave, the OFF responses permit a more sensitive evaluation of retinal electrophysiology.


Orbital meningoencephalocele in a healthy adult.

A 28-year-old white female had a two month history of a slightly painful right eye without proptosis. A radiolucent area was noted in the roof of the right orbit. Orbital exploration demonstrated brown friable tissue without any communication to the cranium. Microscopic examination revealed brain tissue, glial cells and fibrous tissue compatible with dura.


Symposium on diarrhea. 1. Definition and mechanisms of diarrhea.

Diarrhea, an increase in frequency of evacuation and in water content of the stool, is the result of three categories of mechanism--solute malabsorption, secretion of fluid and motility disturbance. Before diarrhea is considered an abnormal condition, any alteration in stool frequency and content must be related to an individual person's normal bowel habit and to norms for the population, but more than three bowel movements or the passage of liquid stools exceeding 300 g daily should, in general, be considered abnormal. A useful way of understanding the mechanism of diarrhea is to become familiar with the normal functions of the bowel in regard to water and electrolyte absorption and motility, and then to relate these functions to solute malabsorption, fluid secretion and motility disturbance.


Symposium on diarrhea. 2. Clinical diagnosis of the causes of diarrhea.

Elucidation of the cause of diarrhea is facilitated by considering which of three principal determinants are of relevance in a particular case. These determinants relate to the onset (whether diarrhea is acute or chronic), to infection or an absence ofinfection and to the presence in the stool of blood or mucus, or both. Diagnosis is also facilitated by taking an accurate and full history, conducting a physical examination, performing sigmoidoscopy and, with proper care, attempting a therapeutic diagnosis. Findings from investigations should then enable one to arrive at a diagnosis. The diagnosis can be reached in an orderly fashion by classifying the types of diarrhea into eight categories: with respect to the acute or chronic onset a case of diarrhea may be noninfectious, without blood or mucus in the stool; noninfectious, with blood or mucus, or both; infectious, without blood or mucus; and infectious, with blood or mucus in the stool.


Symposium on diarrhea. 4. Diarrhea in the irritable colon syndrome.

The irritable colon syndrome comprises two predominant symptom patterns -- "spastic colon" with pain and constipation, and painless "nervous diarrhea". The two patterns frequently overlap. Low intake of dietary fibre is common to patients in both groups. Diagnosis of the irritable colon as a cause of diarrhea requires the characteristic symptom pattern and exclusion of organic disease. Management is based on common sense, careful reassurance of the patient, detailed explanation of the symptom pattern and explicit dietary advice. Increasing fibre in the diet is of prime importance in most patients.


Symposium on diarrhea. 5. Postoperative diarrhea.

Under normal circumstances many physiologic mechanisms prevent diarrhea. The most important are the normal processes of gastric emptying, digestion and absorption, the functioning of the ileocecal valve and elimination of bacteria from the small bowel. A surgical procedure may disrupt any of these mechanisms or may unmask a previously asymptomatic disorder that now requires appropriate treatment. Treatment of diarrhea related to surgery may be directed toward the diarrhea itself with such agents as tranquilizers and opiates but if these are not soon effective any disrupted functions must be analysed and specific measures taken to correct or compensate for these disruptions. The most useful measures in appropriate cases are the prevention of the dumping syndrome and the administration of pancreatic enzymes, cholestyramine, hydrophilic stool thickeners and broad-spectrum antibiotics. These measures may be ineffective or inadequate and surgical revision may be necessary.


Symposium on diarrhea. 6. Infectious diarrhea.

Diarrhea may be primarily infectious in origin. Causes can be conveniently classified according to the etiologic agent, which may be viral, chlamydial, bacterial, protozoal, helminthic or fungal. The most common type of infectious diarrhea in Canada is viral. Bacterial infection, particularly staphylococcal and salmonellal, also is relatively common.