Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows I: Basis for development of protocols

Five experiments were conducted on commercial farms in Brazil aiming to develop a fixed-time artificial insemination (TAI) protocol that achieved pregnancy rates between 40% and 55% in Bos indicus cows. These studies resulted in the development of the following protocol: insertion of an intravaginal device containing 1.9 g of progesterone (CIDR) plus 2.0 mg im estradiol benzoate on Day 0; 12.5 mg im dinoprost tromethamine on Day 7 in cycling cows or on Day 9 in anestrous cows; CIDR withdrawal plus 0.5 mg im estradiol cypionate plus temporary calf removal on Day 9; TAI (48 h after CIDR withdrawal) plus reuniting of calves with their dams on Day 11. Reduced dose of prostaglandin F_2α (PGF_2α; 12.5 mg im dinoprost tromethamine) effectively caused luteolysis. In cycling cows, fertility was greater when the treatment with PGF_2α was administered on Day 7 than on Day 9, but in anestrous cows, no effects of time of the PGF_2α treatment were found. Estradiol cypionate effectively replaced estradiol benzoate or gonadotropin-releasing hormone as the ovulatory stimulus, reducing labor and cost. In this protocol, CIDR inserts were successfully used four times (9 d each use) with no detrimental effects on fertility.     

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.     

Interaction of poly(l-lysine) and Ca2+ with stearic acid monolayers

Interaction of poly(l-lysine) and Ca²⁺ with stearic acid monolayers is studied at pH 9.1, 9.9 and 10.7. The competition between the condensation effect of Ca²⁺ and the expansion effect of the protein on the monolayer is seen to depend on surface pressure as well as pH. Ca²⁺ is much less effective in the competition when the poly(l-lysine) penetration into the monolayer is stabilized by electrostatic interactions.     

Mutagenesis and transformation of C3H 10T12 cells treated with ethyl methanesulfonate

Survival, mutagenesis and transformation were measured in mouse embryo C3H $\text{10}\text{T}\text{1}\text{2}$ cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.     

Association between the cytotoxicity of thymidine and tumorigenicity of clones derived from C3H10T12 mouse embryo fibroblasts

We have measured the cytotoxicity of thymidine to $\text{C3H}\text{10T}\text{1}\text{2}$ mouse embryo fibroblasts derived from morphologically transformed foci of cells from cultures exposed to chemical carcinogens. Four of these cell lines have previously been shown to be tumorigenic in irradiated syngeneic hosts and were all more sensitive to the lethal effects of thymidine than were the non-transformed cells. Strikingly, the most tumorigenic of the cell lines were most sensitive to thymidine. Differences in plating efficiencies or growth rates of the various cell lines were not associated with differences in thymidine sensitivity.     

Mechanically activated transformations in the coordination sphere of platinum complexes induced by impact grinding of solid K2PtX6 (X=Cl, Br) and K2PtCl4 salts

Mechanical treatment of solid K₂PtX₆ (X=Cl, Br) salts under air or argon leads to the formation of paramagnetic platinum(III) complexes via homolytic cleavage of Pt---X bonds. Lewis acid sites (LASs) were also detected on the surface of mechanically activated K₂PtCl₆ using a paramagnetic probe. The latter species can be attributed to coordinatively unsaturated platinum(IV) complexes formed as a result of heterolysis of Pt---Cl bonds. Mechanical treatment of solid K₂PtCl₄, on the contrary, does not lead to homolytic Pt---Cl bond cleavage. In this case only heterolysis of the Pt---Cl bond takes place, leading to the formation of coordinatively unsaturated platinum(II) complexes.     

Coordination polymers of Cu(II) and Cd(II) with bifunctional chelates of the type dipicolylamino-alkylcarboxylate, (NC5H4CH2)2N(CH2)nCO2H (n = 1, 2, 3 and 4)

A series of bifunctional chelates of the type dipicolylamino-alkylcarboxylate (NC₅H₄CH₂)₂N(CH₂)_nCO₂H (n = 1-4; HL¹-HL⁴, respectively) has been prepared. Reactions of the ligands in aqueous methanol/N,N-dimethylformamide with the appropriate Cu(II) salts yielded the compounds [CuL¹](NO₃)·H₂O (1·H₂O), [CuL²(H₂O)]BF₄·H₂O (2·H₂O), [Cu(HL³)(SO₄)]₂ (3) and [CuL⁴(NO₃)]·MeOH (4·MeOH). While compounds 1, 2 and 4 are one-dimensional, the detailed connectivities within the chains are quite distinct, depending on factors such as alkyl chain length and ligation of aqua ligands or anionic components. In contrast to 1, 2 and 4, the structure of 3 is molecular, a binuclear assembly of edge-sharing Cu(II) ‘4+2’ distorted octahedra. The Cd(II) species, [{CdL²}₂(SO₄)]·4H₂O (5·4H₂O), prepared from HL² and CdSO₄·nH₂O in aqueous methanol/N,N-dimethylformamide, is two-dimensional, with a network constructed from binuclear units of seven coordinate Cd(II), , linked through bridging SO₄²⁻ groups to produce an assembly of linked hexagonal rings [{CdL²}₂(SO₄)]₆.     

Comparison of heart rate monitoring combined with indirect calorimetry and the doubly labelled water (2H218O) method for the measurement of energy expenditure in children

The aim of the present study was to compare data on 24-h energy expenditure (EE24h) in nine boys and ten girls (mean age 9.3 and 8.1 years, respectively) by heart rates (fc) combined with energy expenditure obtained from a 1-day stay in an indirect calorimeter (EEcal) and a 2-week period of normal living using the doubly labelled water method (EEdlw). Individual calibration curves were derived from fc and oxygen uptake measured during sleep (in the calorimeter), standing and walking on a treadmill. An estimation of energy expenditure based on 24-h fc monitoring (EEfc) was made during the stay in the calorimeter and on a normal school-day. Mean results showed an overestimation in EEfc compared to EEcal and EEdlw of 10.4% and 12.3% respectively, varying from 6.3% to 16.2%. These results confirmed earlier observations in adults that for a group the fc method overestimates EE24h by about 10%.     

Effects of 2-deoxy-d-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 10⁷ cells/ml. ³¹P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by ¹³C- and ¹H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-¹³C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.     

Synthesis and degradation of cyclic nucleotides in the pituitary gland,ovary and testis of rats treated with d-Trp6-luteinizing hormone-releasing hormone

The effect of administration of d-Trp⁶-Luteininzing Hormone-Releasing Hormone (LH-RH) on synthesis and degradation of cyclic nucleotides was studied in the rat. There were no significant changes in the rate of synthesis and degradation of cyclic AMP in the ovary, testis and pituitary gland of d-Trp⁶ LH-RH-treated rats as compared to controls. On the other hand, the levels of cyclic GMP and activity of guanylate cyclase were significantly higher in the ovary and testis as well as in the pituitary gland of animals which received the analog. The rate of hydrolysis of cyclic GMP was unchanged by the administration of d-Trp⁶-LH=RH. Interestingly, the cyclic CMP phosphodiesterase seemed to be activated in animals treated with d-Trp⁶-LH-RH.     

Reactions of the haloalcohols HO(CH2)nCl (n = 2, 3) with platinum(II) - alkynyl and -alkyne complexes. X-ray crystal structure of trans-[Pt(Me) {C(OCH2CH2Cl) (CH2Ph)} (PPh3)2] [BF4]

The chloro complexes trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄, react with 1-alkynes HC---C---R in the presence of NEt₃ to afford the corresponding acetylide derivatives trans-[Pt(Me) (C---C---R) (PPh₃)₂] (R = p-tolyl (1), Ph (2), C(CH₃)₃ (3)). These complexes, with the exception of the t-butylacetylide complex, react with the chloroalcohols HO(CH₂)_nCl (n = 2, 3) in the presence of 1 equiv. of HBF₄ to afford the alkyl(chloroalkoxy)carbene complexes trans-[Pt(Me) {C[O(CH₂)_nCl](CH₂R) } (PPh₃)₂][BF₄] (R = p-tolyl, N = 2 (4), N = 3 (5); R=Ph, N = 2 (6)). A similar reaction of the bis(acetylide) complex trans-[Pt(C---C---Ph)₂(PMe₂Ph)₂] with 2 equiv. HBF₄ and 3-chloro-1-propanol affords trans-[Pt(C---CPh) {C(OCH₂CH₂CH₂Cl)(CH₂Ph) } (PMe₂Ph)₂][BF₄] (7). T alkyl(chloroalkoxy)-carbene complex trans-[Pt(Me) {C(OCH₂CH₂Cl)(CH₂Ph) } (PPh₃)₂][BF₄] (8) is formed by reaction of trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄ in HOCH₂CH₂Cl, with phenylacetylene in the presence of 1 equiv. of n-BuLi. The reaction of the dimer [Pt(Cl)(μ-Cl)(PMe₂Ph)]₂ with p-tolylacetylene and 3-chloro-1-propanol yields cis-[PtCl₂{C(OCH₂CH₂CH₂Cl)(CH₂C₆H₄-p-Me}(PMe₂Ph)] (9). The X-ray molecular structure of (8) has been determined. It crystallizes in the orthorhombic system, space group Pna2₁, with a = 11.785(2), B = 29.418(4), C = 15.409(3) Å, V = 4889(1) ų and Z = 4. The carbene ligand is perpendicular to the Pt(II) coordination plane; the PtC(carbene) bond distance is 2.01(1) Å and the short C(carbene)-O bond distance of 1.30(1) Å suggests extensive electronic delocalization within the Pt---C(carbene)---O moietry.     

Studies of the reaction products of adenosine with [Pt(NH3)3Cl]Cl and cis-Pt(NH3)2Cl2 by high performance liquid chromatography

The reaction products of adenosine with [Pt(NH₃)₃Cl]Cl or cis-Pt(NH₃)₂Cl₂ have been studied using high performance liquid chromatography and uv spectroscopy. The reaction of [Pt(NH₃)₃Cl]Cl with adenosine (pH = 7.0, Pt/base = 0.5) gives four products. Two of them, mononuclear complexes in which platinum is bound to adenosine through N(7) or N(1), comprise more than 90% of all the products. The N(1) and N(7) sites on adenosine indicate almost equal binding affinity for [Pt(NH₃)₃Cl]Cl. The reaction of cis-Pt(NH₃)₂Cl₂ with adenosine has been studied in the presence of a large excess of adenosine (Pt/base ? 0.05). The reaction gives four products. One is the monomeric 2:1 complex with cis-Pt(NH₃)₂²⁺ bound to two adenosine molecules through the N(7) site and the N(1) site, and another is the monomeric 2:1 complex with cis-Pt(NH₃)₂²⁺ bound to two adenosine molecules through the N(7) sites. cis-Pt(NH₃)₂Cl₂ is stronger affinity to the N(7) site than of adenosine to the N(1) site.     

Inhibition of the glucose and amino-acid carriers of Lemna gibba by pretreatment with HgCl2

The electrical resting potential across the plasmalemma of Lemna gibba L. (G 1) cells is −230 to −250 mV and the diffusion potential in the presence of 1 mol m⁻³ KCN + 1 mol m⁻³ salicylhydroxamic acid is about −100 mV. A concentration of 0.01 mol m⁻³ HgCl₂ depolarises the transmembrane electrical potential in a largely reversible way. When the cells after 16 min of HgCl₂-application are returned to Hg-free solution, the transmembrane electrical potential is only depolarised by 24 × 13 mV (SD, n = 13) compared with the potential prior to HgCl₂ treatment. In contrast, a 16 min pretreatment with HgCl₂ followed by a wash with mercury-free solution reduces the transient depolarisations of transmembrane potential observed after addition of 5 mol m⁻³ D-glncose or 1 mol m⁻³ L-alaoine to about 60% of controls. These transient depolarisations are due to the onset of solute uptake. Accordingly, HgCl₂-pretreatment inhibits uptake of ¹⁴C-3-O-methyl- d -glucose by more than 50% and uptake of ¹⁴C- l -alanine by more than 70%. Washing with 1 mol m⁻³ 1,4-dithiothreitol does not reverse this inhibition. It is, therefore, concluded that Hg²⁺ irreversibly binds to essential SH-groups of the H⁺-hexose and the H⁺-amino-acid cotransport carriers of Lemna gibba and inhibits these carriers without appreciably affecting the electrogenic proton-extrusion pump.     

Effects of CO2 and nitrogen supply on the biochemical composition of Ulva rigida with especial emphasis on lipid class analysis

Lipid class composition was analysed in the green macroalga Ulva rigida grown under normal (350 ppm) and high (10,000 ppm) CO₂ levels, and in nitrate saturated and nitrogen limited conditions. A new protocol for the extraction of lipids has been defined. Culture conditions altered the fate of assimilated carbon, and significant changes were observed in protein and total lipid content in particular. A CO₂-enriched atmosphere conditioned the effects of nitrogen limitation on lipid class composition, revealing deep qualitative changes in carbon metabolism. Triglycerides accumulated at high CO₂ and under nitrogen limitation, while chloroplast-related lipids showed an inverse response. Changes in phospholipids could be related to carbon availability as they did not respond to nitrogen limitation. The ratio sterols/acetone-mobile polar lipids followed a negative linear relation with the optimum quantum yield for photosynthetic electron transport (F_v/F_m), and was considered as an index of the «light status» of the cell. The specificity of the response of lipid classes to growth conditions in U. rigida emphasizes the potential role of lipid class analyses as a diagnostic tool for environmental stress.     

Heats of reaction of HMo(CO)3(C5R5) (R = H, CH3) with diphenyldisulfide and of formation of the clusters [PhSMo(CO)x(C5H5)]2, X = 1,2. Thermodynamic study of molybdenum-sulfur bond strengths

The enthalpies of reaction of HMo(CO)₃C₅R₅ (R = H, CH₃) with diphenyldisulfide producing PhSMo(CO)₃C₅R₅ and PhSH have been measured in toluene and THF solution (R = H, ΔH= −8.5 ± 0.5 kcal mol⁻¹ (tol), −10.8 ± 0.7 kcal mol⁻¹ (THF); R = CH₃, ΔH = −11.3±0.3 kcal mol⁻¹ (tol), −13.2±0.7 kcal mol⁻¹ (THF)). These data are used to estimate the Mo---SPh bond strength to be on the order of 38–41 kcal mol⁻¹ for these complexes. The increased exothermicity of oxidative addition of disulfide in THF versus toluene is attributed to hydrogen bonding between thiophenol produced in the reaction and THF. This was confirmed by measurement of the heat of solution of thiophenol in toluene and THF. Differential scanning calorimetry as well as high temperature calorimetry have been performed on the dimerization and subsequent decarbonylation reactions of PhSMo(CO)₃Cp yielding [PhSMo(CO)₂Cp]₂ and [PhSMo(CO)Cp]₂. The enthalpies of reaction of PhSMo(CO)₃Cp and [PhSMo(CO)₂Cp]₂ with PPh₃, PPh₂Me and P(OMe)₃ have also been measured. The disproportionation reaction: 2[PhSMo(CO)₂Cp]₂ → 2PhSMo(CO)₃Cp + [PhSMP(CO)Cp]₂ is reported and its enthalpy has also been measured. These data allow determination of the enthalpy of formation of the metal-sulfur clusters [PhSMo(CO)_nC₅H₅]₂, N = 1,2.     

Metal carbonyls of telluroether formed by the oxygen atom transfer reaction to M(CO)6 (M = Mo, W) in the presence of (p-CH3OC6H4)2TeO and relative kinetic investigation

A new synthetic process is reported for the preparation of two substituted metal carbonyls, (p-CH₃OC₆H₄)₂TeM(CO)₅ (M = Mo, W). In the presence of (p-CH₃OC₆H₄)₂TeO as O atom transfer reagent in tetrahydrofuran solvent, a CO ligand is replaced by telluroether when M(CO)₆ (M = Mo, W) is reacted with (p-CH₃OC₆H₄)₂TeO under very mild experimental conditions (r.t.). The products were characterized by elemental analysis, mass, IR and ¹H NMR spectroscopies. The spectra suggest that the coordination geometry is distorted from a regular octahedral structure due to an asymmetrical bulky telluroether ligand on the metal atom. Kinetics of these reactions of M(CO)₆ with (p-CH₃OC₆H₄)₂TeO show the reactions are first order in the concentration of M(CO)₆ and of Te oxide. The rates of reaction decrease in the order W(CO)₆>Mo(CO)₆>Cr(CO)₆, and the results obtained are discussed in term of a presumed mechanism.     

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment—the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.     

Theoretical reconsiderations when estimating the mesophyll conductance to CO2 diffusion in leaves of C3 plants by analysis of combined gas exchange and chlorophyll fluorescence measurements

Existing methods to estimate the mesophyll conductance to CO₂ diffusion ( g _m) are often based on combined gas exchange and chlorophyll fluorescence measurements. However, estimations of average g _m by these methods are often unreliable either because the range of usable data is too narrow or because the estimations are very sensitive to measurement errors. We describe three method variants to estimate g _m, for which a wider range of data are usable. They use curve-fitting techniques, which minimise the sum of squared model deviations from the data for A (CO₂ assimilation rate) or for J (linear electron transport rate). Like the existing approaches, they are all based on common physiological principles assuming that electron transport limits A . The proposed variants were far less sensitive than the existing approaches to 'measurement noise' either created randomly in the generated data set or inevitably existing in real data sets. Yet, the estimates of g _m from the three variants differed by approximately 15%. Moreover, for each variant, a stoichiometric uncertainty in linear electron transport-limited photosynthesis can cause another 15% difference. Any estimation of g _m using gas exchange and chlorophyll fluorescence measurements should be considered with caution, especially when g _m is high.     

Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis

Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A₂ from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca²⁺-independent membrane damaging activity against liposome membranes were compared. Residues 116-119 and 122-125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.     

Investigation of intermolecular hydrogen bond interactions in crystalline l-Cysteine by DFT calculations of the oxygen-17, nitrogen-14, and hydrogen-2 EFG tensors and AIM analysis

A systematic computational study is carried out to investigate hydrogen bond (HB) interactions in the real crystalline structures of l-Cysteine at 30 and 298 K by density functional theory (DFT) calculations of electric field gradient (EFG) tensors at the sites of O-17, N-14, and H-2 nuclei. One-molecule (monomer) and nine-molecule (cluster) models of l-Cysteine are created by available crystal coordinates at both temperatures and the EFG tensors are calculated for both models to indicate the effect of HB interactions on the tensors. The calculated EFG tensors at the level of B3LYP and B3PW91 DFT methods and 6-311++G?? and cc-pVTZ basis sets are converted to those experimentally measurable nuclear quadrupole resonance (NQR) parameters i.e. quadrupole coupling constants (qcc) and asymmetry parameters (η_Q). The evaluated NQR parameters reveal that the EFG tensors of ¹⁷O, ¹⁴N, and ²H are influenced and show particular trends from monomer to the target molecule in the cluster due to the contribution of target molecule to classic N–H…O, and non-classic S–H…O and S–H…S types of HB interactions. On the other hand, atoms in molecules (AIM) analyses confirm the presence of HB interactions and rationalize the observed EFG trends. The results indicate different contribution of various nuclei to HB interactions in the cluster where O2 and N1 have major contributions. The EFG tensors as well as AIM analysis at the H6 site show that the N1-H6…O2 HB undergoes a significant change from 30 to 298 K where changes in other N–H…O interactions are almost negligible. There is a good agreement between the calculated ¹⁴N NQR parameters and reported experimental data.