Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

Studies on lectins XL. O-glycosyl derivatives of spheron in affinity chromatography of lectins

Free monosaccharides can be used for direct glycosylation of Spheron, a spherical macroporous hydroxyyalkyl methacrylate-ethylene dimethacrylate copolymer, in a reaction that proceeds at room temperature in dioxane medium under catalysis of dry HCl or BF₃. Derivatives of L-fucose, D-galactose, D-glucose, D-mannose, $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{galactosamine}$ and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ thus prepared from Spheron beads have been shown to be efficient affinity carriers in isolation of lectins from seeds of Canavalia ensiformis D.C. (concanavalin A), Dolichos biflorus L., Glycine soja (L.) Sieb. et Zucc., Lens esculenta Moench, Ricinus communis L., Ulex europaeus L. and from albumin glands of the garden snail Helix pomatia L.     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

Pharmacological properties of imidazole. 2. Action on the normal body temperature regulation in rats

Rats injected intraperitoneally with imidazole (100 to 400 mg/kg) showed a progressive dose-dependent decrease in rectal temperature. A decrease of 6$\text{°}$C occurred when the imidazole-treated animals were placed in a 4$\text{°}$C environment. In a 23$\text{°}$C environment, the rectal temperature declined about 2$\text{°}$C. The results show that imidazole induces a decline in body temperature in non-febrile rats, possibly due to an action on hipothalamic calcium levels.     

Topography of nucleic acid helices in solutions. 28. Evidence for a dynamic structure of DNA in solution

Proton magnetic resonance (pmr), ultraviolet absorption, induced circular dichroism (CD), and viscometric evidence is presented which show that reporter molecules $\text{1}$ and $\text{2}$ bind to DNA via an intercalation process. Preliminary kinetic studies show that the DNA·$\text{1}$ complex forms rapidly (i.e., <1 msec), whereas the DNA·$\text{2}$ complex forms at a considerably slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 100 msec}$). The kinetic results, and the steric requirements for intercalation of $\text{2}$ can be explained on the basis of a dynamic structure of DNA.     

Structural investigations of mode of action of drugs. I. Molecular structure of mitomycin C.

The crystal and molecular structure of the antitumor antibiotic mitomycin C has been determined by X-ray diffraction. The space group is monoclinic P2₁ with cell dimensions $\text{a}\text{=77.988(3)}$, $\text{b}\text{=20.355(7)}$, $\text{c}\text{=9.679(3)}\text{A}\text{?}$, β=95.99°(1) and Z=4. The structure was solved by direct methods. There are two independent molecules per asymmetric unit. The structure was refined to an R value of 0.049 for 2151 observed reflections measured on diffractometer. The benzoquinone ring is slightly deviated from planarity. The N4 in the indole ring behaves like an amide nitrogen owing to its participation in the conjugated benzoquinoid system. The five membered ring through C1, C2, C3, N4 and C9a adopts an envelope conformation. The molecules are held together in crystal by the hydrogen bonds. All nitrogens except N4 are involved in the hydrogen bonding. Studies with models of drug and DNA indicate two different kinds of mechanism for crosslinking.     

Reversed-phase,ion-pair liquid chromatography of quaternary ammonium compounds : Determination of pyridostigmine,neostigmine and edrophonium in biological fluids

A reversed-phase, ion-pair liquid chromatographic method for the quantitative determination of quaternary acetylcholinesterase inhibitors is described. The method uses an ion-pair extraction to isolate the drugs from biological material prior to liquid chromatographic separation and online UV detection at 214 nm. Quantitation down to 5 ng/ml and within-day precision with coefficient of variation (C.V.) of 1.5% (n=10, $\text{x}$ = 100 ng/ml) for neostigmine, C.V., 1.7% (n=10, $\text{x}$ = 80 ng/ml) for pyridostigmine and C.V., 1.5% (n=10, $\text{x}$ = 100 ng/ml) for edrophonium have been achieved. The assay was designed for pharmacokinetic studies of these drugs in anesthetized patients.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

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.     

Uterine infections in the postpartum cow: II. Possible synergistic effect of Fusobacteriumnecrophorum and Corynebacteriumpyogenes

Clinical conditions, which were observed in primiparous Angus and Hereford heifers with postpartum uterine infections are reported. Forty-three of sixty-four cows (67%) had uterine infections. $\text{Corynebacterium}$$\text{pyogenes}$ and $\text{Fusobacterium}$$\text{necrophorum}$ were the most frequently isolated aerobe and anaerobe, respectively. Twelve of the sixty-four cows (18.8%) had infections that involved these species. Three of these twelve cows were infected only with $\text{C.}$$\text{pyogenes}$, two were infected only with $\text{F.}$$\text{necrophorum}$, and seven were infected with both organisms. All five of the cows which were infected with either $\text{C.}$$\text{pyogenes}$ or $\text{F.}$$\text{necrophorum}$ showed signs of estrus and four of the five cows conceived by 110 days postpartum. The single cow that did not conceive was infected with $\text{C.}$$\text{pyogenes}$. Three of the seven cows which were infected with both organisms showed signs of estrus and none of the seven cows conceived by 110 days postpartum. In addition, when only $\text{C.}$$\text{pyogenes}$ or $\text{F.}$$\text{necrophorum}$ was isolated from the uterus, cows had either mild or no clinical signs of infection. In contrast, the seven cows which were infected with both organisms had severe clinical signs of infection that included excessive vulvar discharge, uterine abscesses and pelvic adhesions. These observations suggested that a pathogenic synergism between $\text{C.}$$\text{pyogenes}$ and $\text{F.}$$\text{necrophorum}$ might have caused the increased severity of postpartum uterine infections, and the subsequent detrimental effect on return to estrus and conception.     

Pyrenesulfonyl azide: a covalent probe permitting in vitro desensitization of labeled acetylcholine-rich membrane fragments from Torpedo californica.

Acetylcholine receptor-rich membrane fragments from $\text{Torpedo}\text{californica}$ electroplax after covalent labelling at the protein-lipid boundary by nitrenes generated $\text{in}\text{situ}$ from pyrenesulfonyl azide can bind $\text{[}{}^{125}\text{I]-α-bungarotoxin}$. The covalent attachment of 6–8 molecules of the fluorescent probe/receptor molecule also does not perturb the marked effect on the rate of α-bungarotoxin binding to electroplax membranes exerted by their preincubation with carbamylcholine. This phenomenon, which is analogous to pharmacological desensitization of receptors in synaptic junctions, is fully reversible upon removal of carbamylcholine (Quast, V., Schmerlik, M., Lee, T., Witzemann, V., Blanchard, V. and Raftery, M.A. (1978) Biochemistry $\text{17}$, 2405–2414). Torpedo electroplax membranes, whether tagged with the covalent probe or freshly isolated, regain the original fast rate of α-bungarotoxin binding upon dilution of carbamylcholine.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Selective beta-adrenoceptor activities of tetrahydronaphthalene derivatives.

$\text{Alpha}$- and $\text{beta}$-adrenoceptor activities of a group of tetralin derivatives prepared as fixed analogs of isoproterenol, adrenaline and noradrenaline were evaluated $\text{in vitro}$ assays. All of the present compounds showed strong direct β-stimulating activity fairly selective for tracheal muscle. Activity of a $\text{trans}$ isomer about the C(1)-C(2) bond was ten times higher than that of the $\text{cis}$ isomer. None of the compounds tested had any practical α-adrenoceptor activity. Structure-activity interrelationships between the present series of compounds and some known adrenergic agonists are discussed.     

Outer membrane of gram-negative bacteria. XVII. Secificity of transport process catalyzed by the lambda-receptor protein in Escherichia coli.

The outer membrane of Gram-negative bacteria contains (a) “porin” proteins that form transmembrane channels and allow diffusion of various hydrophilic, small molecules (Nakae, J. Biol. Chem., 251, 2176–2178, 1976), and (b) proteins which catalyze the specific transport of unique classes of compounds, e.g. the λ-receptor protein facilitates the diffusion of maltose and maltotriose (Szmelcman et al., Eur. J. Biochem., 65, 13–19, 1976). When strains of $\text{Escherichia coli}$, $\text{B}\text{r}$ and K-12 containing the λ-receptor but not porin were constructed and compared with those containing neither of them, it was found that in the former strains the transmembrane diffusion of glucose and lactose, but not of histidine and 6-aminopenicillanic acid, was significantly accelerated. These results suggest that λ-receptor may facilitate the diffusion of sugars other than maltose.     

Biological membranes are rich in low-frequency motion

Using ¹³C cross-polarization NMR techniques, we have found that the effect of protein on the dynamics of the hydrocarbon interior of a series of biological membranes is to depress the intensity of motion on the nanosecond timescale (i.e., $\text{T}{}_1$ becomes longer) and to enhance the intensity of motion on the timescale of tens of microseconds (i.e., $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ becomes shorter).     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Behavioral fever in anuran amphibian larvae.

Following intraperitoneal injection with killed gram-negative bacteria, $\text{Aeromonas}$$\text{hydrophila}$, tadpoles of $\text{Rana}$$\text{catesbeiana}$ and of $\text{R}$. $\text{pipiens}$ showed significant mean increases in preferred temperature of 2.6°C and 2.7°C, respectively, in an electronic thermoregulatory shuttlebox device. This “behavioral fever” is similar to elevations in preferred temperature previously demonstrated for fishes, reptiles and mammals, although both normal and febrile thermal preferenda vary among vertebrates.