A physicochemical study of the interaction of phosphatidylinositol with buprenorphine and naloxone

The interactions of two opioid molecules (buprenorphine and naloxone) with phosphatidylinositol and phosphatidylcholine were studied in lipid monolayers at the air-water interface. The influence of Na⁺, Ca²⁺, and Mn²⁺ ions in these interactions has also been determined. Neither buprenorphine nor naloxone influence the ordered state of phosphatidylcholine monolayers. On the contrary, both opioid molecules interact specifically with phosphatidylinositol monolayers. The area/molecule of phosphatidylinositol spread on buprenorphine containing subphases is highly affected by this molecule and also by ions. The phosphatidylinositol/naloxone interactions are rather weak and less affected by ions.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - Bup Buprenorphine - Nx naloxone - ODS octadecyl silica - k capacity factor - logP partition coefficient between octanol and water     

Heteroassociation of Caffeine with the Antibiotic Actinocyl- bis(3-dimethylaminopropylamine) in Aqueous Solution: 1H NMR Analysis

The heteroassociation of caffeine (CAF) and the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) (ACT) was studied in aqueous solution by one- and two-dimensional ¹H NMR spectroscopy at 500 MHz. The equilibrium reaction constants, thermodynamic parameters (H and S) of ACT heteroassociation with CAF, the limiting values of proton chemical shifts of their molecules in the heteroassociation complex, and the spatial structure of the ACT–CAF complex were determined from the experimental dependences of proton chemical shifts of the aromatic molecules on concentration and temperature. The parameters of CAF heteroassociation with the phenoxazone antibiotic actinomycin D and its synthetic analogue ACT were comparatively analyzed and conclusions were made on the crucial role of stacking interactions of the chromophores of CAF and the phenoxazone antibiotics in the formation of the heterocomplexes in aqueous solution.     

Formation,structure, and spectrophotometry of air-water interface films containing rhodopsin

Summary Air-water interface films of cattle rhodopsin and defined lipids are formed without the use of organic solvents by a method in which vesicle membranes consisting of egg phosphatidyl choline and purified rhodopsin are osmotically shocked at the interface. Lipid and protein molecules organize as insoluble films at the interface. The structure of these films varies with the lipid to protein mole ratio of the source vesicle membranes. Electron microscopic observations reveal that films formed with membranes of 1501 mole ratio consist of nonoverlapping, randomly distributed vesicle membrane fragments separated by a lipid monolayer. These membrane fragments exist as single sheets on the water surface and occupy approximately 35% of this surface. Essentially all the rhodopsin molecules at the interface are spectroscopically intact and are contained within the membrane fragments. The visible absorption spectrum of the interface films is identical to that of suspensions of rod disc membranes. Moreover, flash illumination of rhodopsin in air-dried multilayers formed from the interface films results in the formation of a stable MetarhodopsinI intermediate (_max480 nm) which can be fully bleached by increasing the relative humidity of the multilayers or can be photoconverted into rhodopsin and, presumably, isorhodopsin. Furthermore, rhodopsin is chemically regenerable at the air-water interface. Bleached rhodopsin can generate dark rhodopsin at the interface in the presence of 11-cis retinal in the aqueous subphase. Thus, the spectroscopic structure and the chemical regenerability function of rhodopsin in these interface films are indistinguishable from those exhibited by the protein in intact rod disc membranes.     

Influx and efflux of organic acids across the soil-root interface of Zea mays L. and its implications in rhizosphere C flow

The influx and efflux of organic acids across the root-soil interface were investigated in intact, sterile maize (Zea mays L.) roots under a variety of experimental conditions. Under nutrient-sufficient conditions the efflux of organic acids was shown to constitute < 1% of the total C lost across the root-soil interface. Under severe nutrient stress, however, the rates of malate and citrate efflux from the root increased 33 and 12 fold respectively. Influx experiments indicated that roots could not directly reabsorb citrate-Fe³⁺ or other metal complexes from solution. Influx of citrate was observed only at high external citrate concentration ( 1 mM) or from solutions with low ionic strengths. It was postulated that citrate influx is of little importance in a soil environment.     

Lipid-protein surface films generated from membrane vesicles: selfassembly,composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm² film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm². Offprint requests to: H. Schindler     

The 13C chemical shifts of amino acids in aqueous solution containing organic solvents: Application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution

Summary The ¹³C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for ¹³C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the ¹³C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133–2141; Howarth and Lilley (1979) Prog. NMR Spectrosc., 12, 1–40). The pentapeptides studied were assumed to be in a random coil conformation and the measured ¹³C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1–29 amino acid fragment of Nle²⁷ human growth hormone-releasing factor. In both cases, the C chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of -helices.     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Permeability change in transformed mouse fibroblasts caused by ionophores,and its relationship to membrane permeabilization by exogenous ATP

Summary Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [³H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (). The data could be explaine by a threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K⁺, influx of Na⁺ and reduction of preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.     

Alkali ion transport through lipid bilayer membranes mediated by enniatin A and B and beauvericin

Summary Stationary conductance measurements with lipid bilayer membranes in the presence of enniatin A and B and beauvericin were performed. For comparison, some valinomycin systems were investigated. It was found that the conductance in the case of enniatin A and B is caused by a carrier ion complex with a 11 stoichiometry, whereas for beauvericin, a 31 carrier ion complex has to be assumed to explain the dependence of the conductance on carrier and ion concentration in the aqueous phase. The current-voltage curves measured with dioleoyl phosphatidylcholine membranes show a superlinear behavior for the three carriers in the presence of potassium. On the other hand, supralinear current-voltage curves were observed with membranes from different monoglycerides, except for beauvericin. The results obtained with enniatin A and B are in a satisfactory agreement with an earlier proposed carrier model assuming a complexation between carrier and ion at the membrane water interface.The discrimination between potassium and sodium ions is much smaller for the enniatins than for valinomycin. This smaller selectivity as well as the fact that potassium ions cause the highest conductance with lipid bilayer membranes may be due to the smaller size of the cyclic enniatin molecules, which contain 6 residues in the ringvs. 12 in the case of valinomycin. Charge-pulse relaxation studies were performed with enniatin A and B, beauvericin, and valinomycin. For monoolein membranes only in the case of valinomycin, all three relaxations predicted by the model could be resolved. In the case of the probably more fluid membranes from monolinolein (^{9, 12}-C_{18: 2}) and monolinolenin (^{9, 12, 15}-C_{18: 3}) for all carrier systems except for beauvericin, three relaxations were observed.The association rate constantk _R , the dissociation rate constantk _D , and the two translocation rate constantsk _MS andk _s for complexed and free carrier, respectively, could be calculated from the relaxation data. The carrier concentration in the aqueous phase had no influence on the rate constants in all cases, whereas a strong saturation of the association rate constantk _R with increasing ion concentration was found for the enniatins. Because of the saturation,k _R did not exceed a value of 4×10⁵ m ^–1 sec^–1 with 1m salt irrespective of carrier, ion, or membrane-forming lipid.A similar but less pronounced saturation behavior was also observed for the translocation rate constantk _S of the free carrier. The other two rate constants were independent of the ion concentration in the aqueous phase. In the case of the enniatins, the translocation rate constantk _MS was not independent from the kind of the transported ion. In the series K⁺, Rb⁺ and Cs⁺,k _MS increases about threefold. The turnover numbers for the carriers as calculated from the rate constants range between 10⁴ sec^–1 and 10⁵ sec^–1 and do not show a strong difference between the individual carriers. The conductance difference in the systems investigated here is therefore mainly caused by the partition coefficients, which are smaller for the enniatins than for valinomycin.     

Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles

Sequence-specific assignments have been obtained for side chain methyl resonances of Val, Leu and Ile in the outer membrane protein X (OmpX) from Escherichia colireconstituted in 60 kDa micelles in aqueous solution. Using previously established techniques, OmpX was uniformly ²H,¹³C,¹⁵N-labeled with selectively protonated Val-^1,2, Leu-^1,2and Ile-¹methyl groups. The thus labeled protein was studied with the novel experiments 3D (H)C(CC)-TOCSY-(CO)-[¹⁵N,¹H]-TROSY and 3D H(C)(CC)-TOCSY-(CO)-[¹⁵N,¹H]-TROSY. Compared to the corresponding conventional experimental schemes, the TROSY-type experiments yielded a sensitivity gain of about 2 at 500 MHz. The overall sensitivity of the experiments was further enhanced more than two-fold by the use of a cryoprobe. Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the ¹-methyls of Ile. The present approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to about 100 kDa, and adds to the potentialities of solution NMR for de novostructure determination as well as for functional studies, such as ligand screening with proteins in large structures.     

The influence of hollyhock extract administration on testicular function in rats

It has been reported, recently that an aqueous extract from hollyhock flowers (Althaea rosea Cav. varietas nigra) induces weak metabolic changes in rat testes. In the present study, the in vivoinfluence of a methanolic extract was investigated on the metabolism and morphology of the rat testis. To this end, histochemical, morphometric and radioimmunological methods were used. The rats drank the extract at a dose of 100mg/day for 7weeks. The histochemical activities of glucose-6-phosphate dehydrogenase (G6PDH) and ⁵-hydroxysteroid dehydrogenase (⁵HSD) increased significantly statistically in the Leydig cells of the experimental rats in comparison with controls. There were no significant changes in either the diameter of seminiferous tubules or the height of seminiferous epithelium after hollyhock administration. Further, only a small amount of hyperplasia of the interstitial tissue was observed. The morphological and histoenzymatic changes in the Leydig cells indicate that the methanolic hollyhock extract has a direct but small influence on rat testes. The insignificant changes in testicular testosterone and estradiol content suggest that the extract does not disturb steroidogenesis.     

The radioracemization of amino acids by ionizing radiation: Geochemical and cosmochemical implications

A number of optically active amino acids, both in the solid state and as sodium or hydrochloride salts in aqueous solution, have been exposed to ionizing radiation from a 3000 Ci⁶⁰Co -ray source to see if radioracemization might accompany their well-known radiolysis. -Ray doses causing 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization, while aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis showed 5–11% racemization. Amino acid hydrochloride salts in aqueous solution, on the other hand, showed little or no radioracemization accompanying their radiolysis. Both radiolysis, and radioracemization were roughly proportional to -ray dose in the range studied (1–36×10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as aqueous sodium salts are discussed, and the absence of radioracemization for aqueous hydrochloride salts is rationalized. Isovaline, a non-protein amino acid which has been isolated from the Murchison meteorite, contains no -hydrogen atom and is therefore incapable of racemizationvia the chemical mechanisms by which ordinary amino acids racemize. Nevertheless, isovaline suffers radioracemization in the solid state to an extent comparable to that shown by ordinary amino acids, as do its sodium and hydrochloride salts in the solid state. The sodium salt of isovaline in aqueous solution, however, fails to racemize during its radiolysis. Several implicaitons of the newly described phenomenon of radiomization are pointed out for the fields of geochemistry and cosmochemistry.A portion of this research has been described previously at the 144th National Meeting of the American Association for the Advancement of Science, Washington D.C., Feb. 12–17, 1978, at the Fourth College Park Colloquium on Chemical Evolution, University of Maryland, College Park, Maryland, Oct. 18–20, 1978, and at the Carnegie Institution of Washington Conference: Advances in the Biogeochemistry of Amino Acids, Airlie House, Warrenton, Virginia, Oct. 29—Nov. 1, 1978.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase     

The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells

Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K⁺ currents of newborn rat cerebellar granule cells. In whole-cell voltageclamp experiments, cerebellar granule neurons exhibited at least two different K⁺ current components: a first transient component which is similar to an I _A-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 m helothermine reduced the pharmacologically isolated I _A-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC₅₀) of 0.52 m. A comparison between control and nelothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC₅₀ = 0.86 m) but not voltage dependent. No frequency-or use-dependent block was observed on both K⁺ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K⁺ current present in central nervous system neurons.     

Molecular motion of small nonelectrolyte molecules in lecithin bilayers

Summary We have used magnetic resonance spectroscopy, both ESR and¹³C spin relaxation, to measure translational and rotational mobilities and partition coefficients of small nitroxide solutes in dipalmitoyl lecithin liposomes. Above the bilayer transition temperature,T _c, the bilayer interior is quite fluid, as determined from the solutes' rapid rotational and moderately rapid translational motion; the rotational and translational viscosities within the bilayer are _R <1cP and =6–10cP, respectively. and _R are independent of molecular size for all solutes studied, but all were small compared to the size of the phospholipids. , and probably _R , are relatively independent of temperature aboveT _c, but both increase very sharply as temperature is lowered belowT _c; at 32°C, _R increases to 6cP and is greater than 1000 cP. Anisotropy of rotational motion increases gradually as temperature is lowered toT _c, and changes little belowT _c; anisotropy of translational motion was not investigated.¹³C nuclear spin relaxation measurements indicate that translational motion of nitroxide solutes is more rapid in the center of the bilayer than near the polar interface. It takes at least 100 nsec for a solute molecule to cross the bilayer/water interface. We estimate a lower limit of 2 sec/cm for the interfacial resistance to solute diffusion; this result indicates that interfacial resistance dominates permeation across the membrane. The relative solubility, or partition coefficient, is a strong function of solute structure, and decreases abruptly on cooling through the transition temperature. From the partition coefficient and its temperature dependence we calculate the free energy, enthalpy, and entropy of partition. Effects of cholesterol on partition and diffusion coefficients are compatible with the interpretation that bilayers containing cholesterol consist of two phases.     

A lyophilized aqueous extract of<Emphasis Type="Italic"> Maytenus ilicifolia</Emphasis> leaves inhibits histamine-mediated acid secretion in isolated frog gastric mucosa

Lyophilized aqueous extract of Maytenus ilicifolia leaves (LAEMIL) is commonly used in Brazilian folk medicine in the treatment of dyspepsia as well as gastric ulcers. We have investigated the effect and the possible mechanism of action of the LAEMIL on acid secretion in isolated frog gastric mucosa incubated in an Ussing chamber. It was observed that LAEMIL (7–28 mg%) as well as cimetidine (125–4,000 M), a well-known histamine H₂ receptor antagonist, decreased basal acid secretion in a concentration-dependent manner. Similarly to cimetidine (190 M), LAEMIL (21 mg%) also inhibited gastric acid secretion induced by increasing concentrations of histamine (50–800 M). The EC₅₀ values for histamine alone and histamine in the presence of LAEMIL or cimetidine were 94.6 M (71.1–125.9 M), 244.9 M (209.4–286.4 M) and 142.2 M (23.6–855.0 M), respectively. LAEMIL, histamine and cimetidine were effective on acid secretion only when added to the serosal surface of the mucosa. Furthermore, simultaneous addition of LAEMIL and cimetidine at concentrations, per se, ineffective, caused a 16% reduction in the basal acid secretion [from 8.3±0.3 to 6.9±0.2 Eq g^–1 (15 min)^–1, n=4]. Although effects such as inhibition of histamine biosynthesis and/or histamine release can not be ruled out, our data suggest that LAEMIL, like cimetidine, reduces acid secretion in the isolated frog gastric mucosa by antagonising histamine H₂ receptors.Abbreviations LAEMIL Lyophilized aqueous extract of Maytenus ilicifolia leaves - Hist Histamine - Cim Cimetidine     

Effect of size on the energy acquired in species of the fish from a neotropical reservoir,Brazil

In this paper we analysed autotrophic sources of the carbon ( ¹³C) and the trophic position ( ¹⁵N) of Leporinus friderici in the influence area of Corumbá Reservoir, Brazil. We collected samples of muscles of fish from different sizes riparian vegetation, C₄ grasses, zooplankton, periphyton and particulate organic carbon (POC). There were significant differences for the carbon isotope proportion found in muscles of L.friderici in the different size groups analysed. The highest values of ¹³C recorded for middle sized individuals is attributed to the large contribution of C₄ plants in their diet. Small individuals sampled upstream also receive similar contribution from C₄ plants. In contrast the same size group sampled downstream from the reservoir, has a much smaller of C₄ plants. The ¹³C negative character of small individuals from downstream is due to the larger contribution of C₃ plants (except periphyton). At larger sizes we found intermediate ¹³C values. The ¹⁵N proportions we found for each size group were not significantly different, however we found decreasing mean values with increasing size. The trophic level calculated from the dietary data was higher than that found with the ¹³C concentration in the muscle, except for small individuals, when the values were equal.     

High frequency electric fields for trapping of viruses

Summary Combining dielectrophoretic and hydrodynamic forces in micro electrode structures allows enrichment and stable trapping of viruses in aqueous solutions. Fluorescently labelled Influenza and Sendai viruses were collected from solutions of 2^*10⁵ – 2^*10⁸ viruses/l within a few seconds. In the central part of the trap a virus aggregate of about 2–9 m in diameter was formed. This corresponds to a local enrichment of viruses up to a factor of about 1400.     

Refined solution structure of the dimeric N-terminal HHCC domain of HIV-2 integrase

The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1–55, named IN_1–55) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739–746], consists of four -helices and is well defined. Helices 1, 2 and 3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix 3. The orientation of the two monomeric units with respect to each other shows considerable variation. ¹⁵N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN_1–55 shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN_1–55 [Cai et al. (1997) Nat. Struct. Biol., 4, 567–577], where the corresponding residues are positively charged and neutral ones.     

Determination of Malathion and Diazinon Resistance by Sequencing the MdαE7 Gene from Guatemala,Colombia, Manhattan,and Thailand Housefly (Musca domestica L.) Strains

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of E7 gene, which is a member of -esterase gene cluster, is probably playing a role in detoxyfication of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly¹³⁷ to Asp substitution was found in the active center of the product of E7 gene. In malathion (an OP) resistant M. domestica strains Trp²⁵¹ to Ser substitution was identified in the active center of the MdE7. In our research, to understand the allelic diversity of the MdE7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA), and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp¹³⁷, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp¹³⁷ and Trp²⁵¹ residues on their both alleles at these two different positions.