Proteins synthesized in lymphoid cells early after activation by phytohemagglutinin

A double-isotope labeling approach has been employed in an attempt to identify the proteins synthesized by lymphocytes early after stimulation by phytohemagglutinin (PHA). The earliest effect of PHA, within the first hour, was the induction of large aggregates of cellular proteins, which were not dissociated by 1% sodium dodecyl sulfate (SDS) in the absence of β-mercaptoethanol. These aggregates were composed of proteins of molecular weight approximately 70,000, but they did not include PHA. The aggregates were made up of preexisting as well as newly synthesized cellular proteins. Subsequently, within the first 2 hr after the addition of PHA, there was a nonspecific stimulation of protein synthesis. This was followed by the preferential synthesis of several classes of proteins including at least one group of nuclear proteins. The structural changes described here are among the earliest events known to occur within the lymphoid cell after its interaction with PHA.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Catastrophe theory of dopaminergic transmission: a revised dopamine hypothesis of schizophrenia

Mathematical modeling of experimentally observed parameters of dopaminergic neuronal activity suggests the occurrence of multiple equilibrium states in neurons characterized by certain precisely defined properties of the tyrosine hydroxylating system. These equilibria may become unstable under certain conditions and transitions between multiple states are predicted. In addition, modeling of the spatial interactions of dopamine neurons within a neural net leads to domain wall soliton-like solutions of neuronal firing. In the discrete spatial case, these equations are isomorphic to those of the Ising model of phase transitions in lattice spins.The hypothesis is proposed that the occurrence of multiple stable equilibrium states rather than excessive dopaminergic transmission forms the pathophysiological basis of the schizophrenic thought disorder.The model is internally consistent with known clinical effects of drugs such as neuroleptics, reserpine and amphetamine. In agreement with postmortem and other studies, the theory predicts the lack of increased concentrations of dopamine or its major metabolite, homovanillic acid, in brain and cerebrospinal fluid of schizophrenics.The mathematical model is compatible with the theory that postulates an attention deficit as an underlying mechanism of schizophrenic psychosis and allows for a possible genetic heterogeneity of the disease.     

Evidence for seasonal variation in aggressive behaviour by Macaca mulatta

Observations were made on the Macaca mulatta colony on the island of Cayo Santiago over a period that included two reproductive cycles. The frequency of aggressive behaviour was found to fluctuate cyclically as a function of the colony's reproductive state. Males were wounded and died significantly more frequently during the mating season than at any other time of year. Females were wounded about as often during the mating season as during the birth season, and more died during the birth season than during the mating season, although not significantly so. There was no significant difference between sexes in age at death, nor was there a significant departure from expectation of number of deaths per age class. However, mortality among the reproductively mature males was greater than among mature females. It was suggested that differential injury and mortality rates among adult males might be an indirect result of high levels of androgens which could conceivably alter threshold levels for stimuli initiating severe aggressive interactions.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

On the transmission of external pressure fluctuations to the cerebrospinal fluid

A theory has been formulated to explain the manner in which external pressure fluctuations are transmitted to the cerebrospinal fluid (CSF). The theory is based upon a three-compartment model which consists of the cerebral ventricles, the basal cisterns and spinal subarachnoid space, and the cortical subarachnoid space. The external pressure disturbance is represented by a Fourier series summed over the frequency ω. The mathematical analysis leads to a time constant τ which depends upon the compliances of the spinal region and sources of external pressure fluctuations, the rate of CSF absorption and the rate of fluid transfer between compartments. For arterial pulsations where ωτ ? 1, the theory is in accord with the experimental observations that (i) the arterial and CSF pulse waves are nearly identical in shape, and (ii) the amplitude of the CSF pulse wave increases with intracranial pressure. Moreover, it predicts that the amplitude of the wave will be larger in the spinal region than in the ventricles. The theory also accounts for the observation of one per minute pulse waves observed in hydrocephalic patients with decreased absorption rates.     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

The compression-ordering and solubility-disordering effects of high pressure gases on phospholipid bilayers.

Inert gases at high pressure may compress and dissolve in tissue of intact organism to result in narcosis, reversal of the effects of anesthetic agents or hyperexcitability. The effects of 51 and 102 atm of helium, hydrogen, nitrogen, argon, xenon and nitrous oxide on the molecular motion of nitroxide spin-labeled phospholipid-cholesterol bilayers were measured by electron paramagnetic resonance (EPR) techniques. Immediately, application of high pressures of all gases decreased the molecular motion of the fatty acid chains of the membrane phospholipids; the magnitude of ordering was linearly related to the amount of pressure applied. The second effect was an increase in molecular motion of the fatty acid chains which appeared more slowly due to the slow gas diffusion through the column of lipid dispersion. The magnitude of disorder of the phospholipid membrane at equilibrium correlated with the known lipid solubilities of the gases in olive oil as well as with the anesthetic potency of all the gases except xenon. The environment of the spin label became less polar as the gases diffused into the bilayer. The present studies in the phospholipid model membrane show that the net effects of high pressure gases in the lipid phase consist of an initial ordering of the membrane by compression opposed by the ability of the gas molecules to diffuse and dissolve in the lipid bilayers and disorder them. It is thus suggested that the resultant perturbations of the membrane lipid fluidity by high pressure gases may subsequently be transmitted to membrane-bound protein to result in changes that may be associated, in part, with the diverse effects of anesthesia and of the high pressure nervous syndrome (HPNS) observed in deep-sea divers. The model system may be useful in developing gas mixtures which minimize HPNS.     

Complexes of vitamin B6, V interaction of pyridoxal with pyridoxamine in the presence of copper(II)

The interaction of pyridoxal (PL) with pyridoxamine (PM) in the presence or absence of CU(II) has been studied in acidic aqueous solutions. We conclude that a ternary complex is formed prior to the interaction of pyridoxal with pyridoxamine in the presence of Cu(II) ions. Although the rate of interaction of excess PL with the Cu(II)-PM system of composition 1:1 followed pseudo-first-order kinetics, this was not so with composition ratios of PM to Cu(II), greater than unity. The observed rate constant (k_obs) has the following form for Cu(II):PM:PL in the ratio 1:1:10 (or more):

     

The electron transfer system of Pseudomonas aeruginosa: a study of the pH-dependent transitions between redox forms of azurin and cytochrome c551

Fast reaction kinetic experiments on the electron transfer reaction between azurin and cytochrome c₅₅₁ isolated from Pseudomonas aeruginosa confirmed the existence of two redox forms of reduced azurin previously reported. The pH dependence of the amplitudes of the relaxation processes observed in temperature jump experiments indicate that these two redox forms are in pH dependent equilibrium. The pH independence of the overall equilibrium constant indicates that redox active and inactive forms of cytochrome c₅₅₁ may also exist. Evidence that reduced cytochrome c₅₅₁ undergoes a pH transition is given by optical spectrophotometry. The nature of the transition is discussed in the context of recent nmr studies and in terms of the Marcus theory of electron transfer. The metabolic consequences of these transitions are also discussed.     

Effect of sample preparation on analysis of superoxide dismutase activity and isoenzymes

The effect of superoxide dismutase (SOD) activity and isoenzyme pattern of detergents, incubation time, and sonication in the preparation of rat liver samples was investigated. The activity of the manganese form of the enzyme (Mn-SOD) was found to decrease significantly after 4 hr of incubation at room temperature, and activity of the copper, zinc form of the enzyme (Cu, Zn-SOD) was not changed significantly even after 24 hr, although levels were somewhat decreased. Sonication of the sample did not affect Cu, Zn-SOD activity, but total Mn-SOD activity was increased. Addition of detergents did not increase Mn-SOD activity when homogenates were sonicated, indicating that Mn-SOD is not membrane bound. Detergents also had no effect on Cu, Zn-SOD activity. None of the treatments investigated altered the isoenzyme patterns, providing evidence that these isoenzymes are not degradation products.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Molecular interpretation of kinetic-ionic strength effects

A recent and important approach to investigating electron transfer mechanisms of redox proteins has been through kinetic-ionic strength studies. There is, however, significant controversy as to whether such studies (1) yield information regarding the charge (or location) of the electron transfer site or (2) more simply reflect the influence of net or overall protein charge on the electrostatic interactions. A critical analysis using different theoretical approaches is made of our recent work and of the bulk of the published non-physiological small molecule-protein and protein-protein kinetic ionic strength studies; it is concluded that (1) the approximated Bronsted-Debye-Huckel equation can not be used at all for protein redox reactions, (2) irrespective of the theoretical approaches discussed, such studies do not provide information regarding the charge of the electron transfer site, (3) it is the net charge of the reactants that control the electrostatic interactions, (4) both the equation derived by Wherland and Gray and the full Bronsted-Debye-Huckel equation provide reasonably good approximations of net protein charge, (5) pH changes quantitatively modulate net protein charge, and (6) thus, protein redox rates need to be electrostatically corrected if relevant interpretations of kinetic-ionic strength experiments are to be made.     

Frequency and intensity of chick distress calls: Effects on maternal foodcalling in chickens

The food call of broody domestic hens was used to measure maternal response to four frequency components found in chick distress calls (2,3,4 and 5 kHz) and to variations in distress call intensity (0—86 dB). Foodcalling increased significantly with frequency of the pure-tone test pulse; response to a taped distress call occurred between 40 dB and 86 dB intensity with a maximum at 60–65 dB. The results suggest that the mother uses the higher frequency components in recognizing the distress call, but responds maximally within a specific intensity range. The selective advantage of such behaviour is discussed.     

Lack of expression of micronuclear genes determining two different enzymatic activities in Tetrahymena thermophila

Abstract. Several (albeit indirect) lines of evidence indicate that the 2n germline micronucleus of the ciliate protozoans is not expressed during vegetative growth, even though it resides in the same cytoplasm and in close physical proximity to the actively expressed large somatic macronucleus (45n). To test this hypothesis more directly and quantitatively at the level of biochemically assayable specific enzyme activities, special heterokaryon strains of Tetrahymena thermophila have been constructed which carry the wild-type alleles specifying galactokinase ( galA ⁺) and phenylalanine hydroxylase ( tyrC ⁺) in the micronucleus only. The heterokaryons were assayed for these two enzyme activities using in vitro radiometric assays. No galactokinase or phenylalanine hydroxylase attributable to the micronuclear genes was observed in such heterokaryons. These results extend the observation of lack of micronuclear gene expression to at least two specific genes, i.e., galA and tyrC , as well as extending previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis. By conjugating two of these heterokaryons to each other, it is now possible to determine precisely and unambiguously at what point during macronuclear differentiation the genes in the new macronucleus begin to be actively expressed relative to the timing of the extensive molecular changes that accompany the differentiation of the new macronucleus. These heterokaryons thus provide an excellent model system for the investigation of fundamental genetic regulatory mechanisms in operation during the differentiation of an entire eukaryotic genome.     

Abundance and tissue distribution of selenocysteine-containing proteins in the rat

The form and distribution of selenium (Se) in proteins from selected tissues of the rat were studied by measuring 75Se radioactivity in animals provided for 5 months with [75Se]selenite as the main dietary source of Se. Equilibration of the animals to a constant specific activity of 75Se allowed the measurement of 75Se to be used as a specific elemental assay for Se. Skeletal muscle, liver and blood accounted for 73% of the whole-body Se and 95% of the total Se-dependent glutathione peroxidase activity. Over 80% of the whole-body Se was in protein in the form of the selenoamino acid, selenocysteine. All other forms of Se that were measured accounted for less than 3% of the whole-body Se. The Se in protein was distributed in seven subunit sizes and nine chromatographic forms. The Se in glutathione peroxidase accounted for one-third of the whole-body Se. These results show that the main use of dietary Se, as selenite, in rats is for the synthesis of selenocysteine-containing proteins. Furthermore, the presence of two-thirds of the whole-body Se in nonglutathione peroxidase, selenocysteine-containing proteins suggests that there may be other important mammalian selenoenzymes besides glutathione peroxidase.     

The binding of copper ions to daunomycin and adriamycin

Using visible absorption, CD, ¹H nmr, and epr spectroscopy, the Cu(II) binging properties of daunomycin, adriamycin, and N-trifluoroacetyl daunomycin in water and ethanol have been explored. The drugs form two water soluble complexes having Cu-drug stoichiometries of 1:1 and 1:2, and with apparent pK_as of formation of 5.6 and 6.5, respectively. At pH values above ～8, the drugs form insoluble polymeric complexes with Cu(II). Similar species are also observed in ethanol. The structure of the compounds have been interpreted in terms of binding of the deprotonated hydroxyquinone portion of the drug to the copper ion. No evidence for the binding of the amino group on daunosamine was found.     

Oxidation of thymidylate synthase by inorganic compounds

Thymidylate synthase from methotrexate-resistant Lactobacillus casei was rapidly and completely inactivated by low concentrations of permanganate, periodate, or potassium triiodide at 0 degree C. The enzyme was not inactivated to any appreciable extent by iodate, iodide, ferricyanate, iodosobenzoate, or hydrogen peroxide. The inactivation by permanganate was retarded by the substrate 2'-deoxyuridylate and, to a lesser extent, by phosphate. Titration of enzyme activity with permanganate showed that two moles of permanganate were required to completely inactivate one mole of thymidylate synthase.     

The reduction of cytochrome c by iron EDTA-like complexes: implications on charge effect corrections to mediator-protein rate constants

The reduction of cytochrome c by a series of ferrous EDTA-like complexes was analyzed by the relative Marcus and Tunneling theories. All of the rate constants were calculated at infinite ionic strengths by the use of the Wherland-Gray equation to eliminate electrostatic effects. Both of the theories predicted the rate constants quite well for most of the complexes. But, for those complexes that cannot completely fill all the coordination sites of the iron (HEDTA, NTA), the rate constants were anomalously low. This would be consistent with an increased tunneling distance due to an increase in the size of the complex due to hydrogen bonding between the coordinated hydroxide and the solvent, or due to poorer interaction between the protein and the more hydrophobic mediators.