Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

Chromosome polymorphism in populations of the grasshopper Trimerotropis pallidipennis from southern Argentina

Three populations of the grasshopper Trimerotropis pallidipennis from southern Argentina have been studied cytologically. A very characteristic B-chromosome was found in all three. They also showed geographical variability in respect of the presence of pericentric inversions, and the inversion system was found to influence chiasma frequency. The Laguna Blanca population, which is on the hypothetical pathway the species is believed to have followed during its migration from northern to southern latitudes, has the same karyotype composition as the N. American form, with fixed inversions in the 3 largest autosomes and the X-chromosome. Its members have a high total chiasma frequency and a great number of interstitial chiasmata. The Sierra de la Ventana population, situated at the absolute eastern border of the species distribution is highly polymorphic with respect to the presence of inversions in the medium chromosomes. Its members have the lowest total chiasma frequency and a greatly reduced number of interstitial chiasmata. Situated geographically between the other two, the Choele-Choel population has the highest frequency of inversions and many of them are homozygous. Its members have a higher total chiasma frequency than that observed in specimens from Sierra de la Ventana, and a greatly reduced number of interstitial chiasmata, similar to that observed in individuals from the latter population.     

Fast polyspermy block and activation potential : Correlated changes during oocyte maturation of a starfish

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.     

Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats.

The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.     

Fluctuations of an alpha-helix.

Fluctuations in backbone dihedral angles in the α-helical conformation of homopolypeptides are studied based on an assumption that the conformational energy function of a polypeptide consisting of n amino-acid residues can be approximated by a 2n-dimensional parabola around the minimum point in the range of fluctuations. A formula is derived that relates 〈Δθ_iΔθ_j〉, the mean value of the product of deviations of dihedral angles ?_i and ψ_i (collectively designated by θ_i) from their energy minimum values, with a matrix inverse to the second derivative matrix F ,_n of the conformational energy function at the minimum point. A method of calculating the inverse matrix F _n^?1 explicitly is given. The method is applied to calculating 〈Δθ_iΔθ_j〉 for the α-helices of poly(L -alanine) and polyglycine. The autocorrelations 〈(Δ?_i)²〉 and 〈(Δψ_i)²〉 at 300°K are found to be about 66 deg² and 49 deg², respectively, for poly(L -alanine), and 84 deg² and 116 deg², respectively, for polyglycine. The length of correlations of fluctuations along the chain is found for both polypeptides to be about eight residues long.     

Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase.

Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.     

Release of dialkylglycerol from purple membrane phospholipids by phospholipase D

When the major polar lipid of purple membrane, a dialkyl analogue of phosphatidyl glycerophosphate, is treated with phospholipase D under the usual assay conditions for this enzyme, the reaction yields dialkylglycerol and glycerol bisphosphate, i.e. the kind of products that would be expected from a phospholipase C reaction. The effect is seen both in native purple membranes and with the pure phospholipid in the form of liposomes. The specific activity and kinetic parameters Km and Vmax of phospholipase D for the purple membrane phospholipid are similar to those for egg phosphatidylcholine. The presence of phospholipase C impurities in the phospholipase D preparations has been ruled out as an explanation for the above observations. A hypothesis is suggested, taking into account the peculiar headgroup structure of the bacterial lipid, to explain the seemingly anomalous enzyme behavior.     

Kinetics of purple membrane dark-adaptation in the presence of Triton X-100

The kinetics of purple membrane dark adaptation were studied at pH 5 and 7, in the presence and absence of the nonionic detergent Triton X-100. The effect of both sublytic and lytic surfactant concentrations has been considered. Our results show that: (a) dark adaptation is faster at pH 5 than at pH 7, (b) dark adaptation is slower, and of smaller amplitude, in the presence than in the absence of Triton X-100. The data may be interpreted in terms of a simple first-order kinetic model, according to which light-dark adaptation would depend basically on the equilibrium between the 13-cis- and the all-trans-isomers. The experiments also suggest that at pH 5, but not at pH 7, solubilizing surfactant concentrations produce a considerable increase in the velocity of the dark adaptation reaction, perhaps through changes in the microenvironment of a protonable group.     

Modeling the process of problem-solving by associative networks capable of improving the performance

In this paper a model of neural network underlying arithmetic prblem-solving is described. Memory models of procedural memory, semantic memory, and working memory, which are necessary to represent the process of the problem-solving, are constructed within a framework of a model of associative processor, HASP, proposed by one of the authors (Hirai 1983). Performance of the model has been simulated on a digital computer. By memorizing primitive knowledge of addition of two digits such as 6+8=14 in the semantic memory and procedural knowledge for the control of the process of adding in the procedural memory, the model can perform addition of multiple numbers with multiple digits. By making explicit serial associations between consecutive procedural steps, the performance of the model can be impooved, because a current procedural step primes the next one. In addition, if a preceding procedural step is a subset of the next one, merging between the two steps occurs. The performance can be improved about 20% by these priming and merging. By memorizing incorrect procedures, the model can generate four kinds of bugs of addition which were observed in children's performance.This paper is a detailed version of the talk presented in CNN'87, but the model is slightly modified