Complexation of Pr3+ by two different ionophores enhances markedly its transport rate

Transport of Pr³⁺ across phosphatidylcholine vesicles, monitored through ³¹P nmr, is first-order in monensin ($\text{1}$), second-order in etheromycin ($\text{2}$) or in lasalocid A ($\text{3}$). When $\text{1}$ and $\text{2}$ (or $\text{2}$ and $\text{3}$) are incorporated $\text{together}$ in 1:1 ratio into the lipidic phase, transport is faster than with each ionophore alone. For instance, assuming that the complexes $\text{2}$.Pr³⁺.$\text{2}$, $\text{3}$.Pr³⁺.$\text{3}$, and $\text{2}$.Pr³⁺$\text{3}$ are equiprobable, they effect transport at intrinsic relative rates of 1, 2, and 13.5, $\text{i.e.}$ a remarkable synergism is set up.     

Structure and properties of hemocyanins. XII. Electron microscopy of dissociation products of Helix pomatia alpha-hemocyanin: quaternary structure

Electron microscopy of the dissociation products of α-hemocyanin of the snail Helix pomatia reveals two distinctly different conformations of $\text{1}\text{10}$-size molecules: a C(compact)-form at high ionic strength and pH near 8, and an L(loose)-form at low ionic strength and/or higher pH.The size and shape of the C-$\text{1}\text{10}$-size molecules indicate a dissociation of the $\text{1}\text{2}$-size molecules along the (10,9) helical grooves, which have been shown to be boundaries between the morphological units of the cylinder wall (Mellema &; Klug, 1972). The 5-fold collar is distributed evenly among the C-$\text{1}\text{10}$-size molecules.The C → L conformational change is characterized by a drastic loosening of the $\text{1}\text{10}$-size molecules to a flexible cluster of globules, with a concomitant decrease of sedimentation coefficient and increase of intrinsic viscosity and frictional ratio.The $\text{1}\text{20}$-size molecule appears as a flexible, linear chain of seven or eight globules with a diameter of 55 to 60 Å. These globules are inferred to be the minimal oxygen binding units with a molecular weight of about 50,000, linked together by short stretches of the polypeptide chain.Stable intermediate dissociation products, $\text{2}\text{10}$, $\text{3}\text{10}$ and $\text{4}\text{10}$-size molecules, were obtained by intramolecular crosslinking of $\text{1}\text{2}$-size molecules with dimethyl-suberimidate prior to dissociation.     

Solid phase peptide synthesis of alpha-factor, a yeast mating pheromone.

Based on analysis of highly purified preparations of natural $\text{α}$-factor and on the sequence recently reported by others, oligopeptides of the following structures were chemically synthesized by the solid phase method of Merrifield: N-Trp-His-Trp-Leu-Lys-Pro-Gly-G1N-Pro-Met-Tyr-C N-His-Trp-Leu-Lys-Pro-Gly-G1N-Pro-Met-Tyr-C Both synthetic species arrested $\text{a}$ cells in G1, inhibited their DNA synthesis, caused them to elongate markedly, and induced an increase in their adhesivity toward $\text{α}$ cells. Neither synthetic material caused any of these effects in $\text{α}$ cells or in $\text{a}\text{α}$ diploids.     

Structure and properties of hemocyanins. XIV. Reassociation of Helix pomatia alpha-hemocyanin

Helix pomatia α-hemocyanin dissociates within minutes into $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules, on increase of pH in the alkaline region.The rate and final state of reassociation of $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules have been studied, by turbidity measurements and ultracentrifugation, on lowering of pH or on the addition of calcium ions.Reassociation of $\text{1}\text{10}\text{-size}$ molecules proceeds in two phases, with half-times in the order of minutes and one hour, respectively. The slow phase is linked to the disappearance of transitory intermediates, presumably dimers and tetramers of $\text{1}\text{10}$-size molecules.A hysteresis is observed in the final state of pH-dependent and Ca²⁺-dependent dissociation-reassociation.Reassociation of $\text{1}\text{20}$-size molecules depends on the initial (isomeric) state of these molecules. The F(fast sedimenting)-$\text{1}\text{20}$-size molecules reassociate almost completely to whole molecules, whereas the S(slow sedimenting)-form does not reassociate at all.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

Effects of depurination on the fidelity of DNA synthesis.

The effect of depurination of polynucleotide templates on the fidelity of DNA synthesis in vitro has been determined. The fidelity of DNA synthesis with Escherichia coli DNA polymerase I, avian myeloblastosis virus DNA polymerase and human placenta DNA polymerase-β is decreased as a result of depurination of the poly[d(A-T)], poly[d(G-C)]and poly[d(A)]templates. The error rate with poly[d(A-T)]increased from $\text{1}\text{17,500}$ to $\text{1}\text{2100}$ using E. coli Pol I, and from $\text{1}\text{4100}$ to $\text{1}\text{1500}$ using the myeloblastosis virus DNA polymerase. Depurination of poly[d(A)]increased the error rate from $\text{1}\text{21,000}$ to $\text{1}\text{6500}$ using E. coli Pol I, and from $\text{1}\text{19,300}$ to $\text{1}\text{6100}$ using the DNA polymerase-β from human placenta. Depurination of poly[d(G-C)]resulted in an increase in the error rate with E. coli Pol I from $\text{1}\text{9200}$ to $\text{1}\text{2200}$, and with the virus DNA polymerase from $\text{1}\text{2400}$ to $\text{1}\text{1300}$. This misincorporation is shown to be directly proportional to the extent of depurination. Deletion experiments and alkaline sucrose gradient analyses suggest that the incorporation of complementary and non-complementary nucleotides is dependent on polymerization, and occurs in the same newly synthesized product. Kinetic studies and nearest-neighbor analyses indicate that the incorporation of non-complementary nucleotides occurs randomly as single-base substitutions. The nearest-neighbor studies also suggest that any of the four deoxynucleotides can be incorporated opposite apurinic sites. The number of each nucleotide incorporated relative to the number of apurinic sites was determined to be $\text{1}\text{490}$ for dGTP, $\text{1}\text{115}$ for dCTP, $\text{1}\text{2·5}$ for dATP and $\text{1}\text{1·7}$ for dTTP with both the poly[d(A-T)] and poly[d(A)] templates. The frequencies of misincorporation relative to the number of apurinic sites with the poly[d(G-C)]template were $\text{1}\text{230}$ for dATP, $\text{1}\text{120}$ for dTTP, $\text{1}\text{2·4}$ for dGTP and $\text{1}\text{1·8}$ for dCTP. Hydrolysis at the apurinic sites by alkali treatment reversed the effects of depurination on fidelity. The error rates with the depurinated templates were reduced to within 2% of those obtained prior to depurination, providing additional evidence that the misincorporation after depurination results from apurinic sites on the template. These results suggest a possible relationship between depurination of DNA and errors in DNA replication and/or repair.     

In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.     

Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium

Cyclic AMP was not found in vegetative cells or sporulating cells or dormant spores of $\text{Bacillus}\text{megaterium}$ using an assay which would have detected an $\text{in}\text{vivo}$ concentration of 1 – 2 × 10^?9 M. Adenyl cyclase and cyclic AMP phosphodiesterase were also not detected in sonicates of vegetative or sporulating $\text{B.}\text{megaterium}$ cells.     

Glucagon or cyclic AMP-stimulated synthesis of 5-phosphoribosyl 1-pyrophosphate in isolated hepatocytes and inhibition by antimicrotubular drugs.

Glucagon increased the level of 5-phosphoribosyl 1-pyrophosphate ($\text{PP}$Rib$\text{P}$) in isolated rat hepatocytes; a relatively high concentration of cyclic AMP could replace glucagon. In the presence of glucagon, the rate of incorporation of respective radioactive precursors into purine, pyrimidine, and oxidized pyridine nucleotides was accelerated, indicating that glucagon stimulates the synthesis of $\text{PP}$Rib$\text{P}$. Addition of 10^?6 M colchicine, vinblastin, or podophyllotoxin abolished the glucagon or cyclic AMP-induced increase in the $\text{PP}$Rib$\text{P}$ level. Colchicine did not affect accumulation of cyclic AMP induced by glucagon. These results suggest the involvement of tubulin or microtubules in the signal transfer from cyclic AMP to stimulated synthesis of $\text{PP}$Rib$\text{P}$.     

Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria - inhibitory effect of puromycin.

$\text{Trans}$-3-dehydro-D, L-ornithine and $\text{trans}$-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of ornithine decarboxylase. The K_I′S for $\text{trans}$-3-dehydro-L-ornithine and $\text{trans}$-1, 4-diamino-2-butene are 2.2 and 2.0 μM respectively. Both analogs bind much more tightly to the enzyme than either ornithine or putrescine. Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of $\text{trans}$-3-dehydro-D, L-ornithine to the culture medium.     

Synthesis of a trans-hydrindane nuclear analog of 11-desoxy-dihydro-prostaglandin E1

Stereosecific synthesis of $\text{trans}$-hydrindanone $\text{2a}$, a bicyclic analog of prostaglandin E₁, $\text{via}$ the $\text{trans}$-hydrindane β-keto ester $\text{8}$, is described. When tested in the guinea pig, $\text{2a}$ exhibited no effects on blood pressure and no broncho-constriction or dilation activity. Additionally, $\text{2a}$ failed to inhibit both ADP $\text{and collagen induced blood platelet aggregation}$.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

Structure and properties of hemocyanins. XIII. Dissociation of Helix pomatia alpha-hemocyanin at alkaline pH

Helix pomatia α-hemocyanin can dissociate stepwise into $\text{1}\text{2}$-size, $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules. Both $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules can occur in two isomeric forms, differing considerably in sedimentation behaviour.The effects of pH, ionic strength, temperature, Ca²⁺ concentration and oxygen pressure on these dissociation and isomerization steps were investigated systematically by sedimentation analysis.Dissociation and isomerization are favoured by increasing pH or temperature. Changes in ionic strength affect each step differently. Calcium ions are extremely effective in preventing dissociation and isomerization at low ionic strength, but this stabilizing ability diminishes at higher ionic strengths. Oxygen binding shifts the pH-dependent dissociation of whole into $\text{1}\text{2}$-size molecules to higher pH. Oxygen has no effect on the other dissociation steps. Intermolecular interactions appear to be predominantly electrostatic.     

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids $\text{1}$, $\text{5}$ and $\text{7}$ with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu₄NOH) and synthesis of 2α-hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ by the controlled hydrolysis of the corresponding 2α-bromo-3-ones $\text{9}$, $\text{12}$ and $\text{15}$ are described. Treatm carbonate in aqueous acetone or with n-Bu₄NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones $\text{3}$, $\text{6}$ and $\text{8}$ in 85–90% yield, respectively. 2α-Hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ were obtained by hydrolysis of the corresponding bromoketones $\text{9}$, $\text{12}$ and $\text{15}$ in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone $\text{9}$ and the 2β-bromo isomer $\text{10}$ precedes the formation of the ketol $\text{11}$ in which the true intermediate might be the 2β-isomer $\text{10}$. However, rearranged androstane derivatives, 3β-hydroxy-2-ones $\text{18}$ and $\text{20}$, were stereoselectively obtained by treatment of the bromoketones $\text{12}$ and $\text{15}$ with an excess amount of sodium hydroxide.     

Inhibition by zinc of the binding of C1 to antigen-antibody complexes

Zinc sulphate in the range of 10^?4 to 2×10^?5 M prevents the binding of $\text{C}\text{1}$ to antigen antibody complexes, and the initation of the cascade of events in the classical complement pathway leading to cell lysis. Other heavy metals, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, or Mn⁺⁺ were without effect in this concentration range. Zinc was ineffective when added after $\text{C}\text{1}$ was bound and failed to displace $\text{C}\text{1}$ which was already bound to antigen antibody complexes. The ability of zinc to regulate the binding of the zymogen or activated form of $\text{C}\text{1}$ to antigen-antibody complexes represents a new method of controlling the initiation of the classical complement pathway.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.