Light-induced changes in cAMP levels in Limulus photoreceptors

Illumination reduces cAMP levels about 3-fold in $\text{Limulus}$ photoreceptors. Cyclic GMP levels are not significantly changed under identical conditions. In addition, the cAMP content in dark-adapted photoreceptors is about 4-fold the content of cGMP. It is proposed that cAMP may be involved in the regulation of the metabolism or function of photoreceptor systems which contain the photopigment in the transducing surface membrane.     

Changes in adrenal dopamine concentration after metyrapone or ACTH administration: implications for the in vivo regulation of dopamine beta-hydroxylase by glucocorticoids.

$\text{In vitro}$ studies have demonstrated that rat adrenal dopamine beta-hydroxylase activity is controlled by neural input and by glucocorticoid production. However, beta-hydroxylation of dopamine $\text{in vivo}$ is a first-order reaction and may be considerably slower than the maximal rate determined by $\text{in vitro}$ methods. To estimate the $\text{in vivo}$ reaction rate the concentrations of dopamine (substrate) and of beta-hydroxylated catecholamine (product) were measured as a function of endogenous glucocorticoid production. Beta-hydroxylated catecholamine changed little but dopamine was increased 2-fold or more 17.5 h after the inhibition of steroidogenesis with metyrapone. Dopamine was also increased by metyrapone in animals with pre-existing adrenal denervation. ACTH 17.5 h before sacrifice caused only slight changes in normal rats but reduced the increase in dopamine caused by stress. The results indicate that adrenal dopamine concentration is inversely related to glucocorticoid production at a given level of neural input and provide $\text{in vivo}$ evidence that glucocorticoids maintain dopamine beta-hydroxylase activity in the adrenal gland.     

The transepithelial shunt pathway in the renal proximal tubule of newt kidney

The transepithelial shunt pathway of newt proximal tubule was examined with glass micro-electrode and electron microscopic methods. The input resistance of the peritubular (basal) membrane and tubular wall were found to be $\text{4.2 ± 0.1 · 10}{}^6$ ($\text{mean ± S.E.}\text{, n = 16}$) and $\text{11.4 ± 0.2 · 10}{}^4\text{(n = 11)}$, respectively. The input resistance of the peritubular membrane was approximately 40-times larger than that of the tubular wall. When the kidneys were perfused in a lanthanum solution, the lanthanum ions were then observed in the junctional complexes and in the intercellular spaces on both the basal and apical sides. The results indicate that the electrical shunt pathway corresponds to the apical junctional complexes and the intercellular spaces, and that the tight junctions are not truly ‘tight’ for the transepithelial movement of small ions in the proximal tubule of the newt kidney.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Enantiomer selection in the nucleophilic addition reaction of optically active amines to α-amino acid N-carboxyanhydrides

The enantiomer selection in the nucleophilic addition reaction of optically active amines such as α-amino acid esters to phenylalanine and $\text{N-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$ in $\text{m-}\text{dimethoxybenzene}$ as a solvent has been investigated. Stereoselectivity between the amines and the $\text{N-}\text{carboxyanhydrides}$ was found to change markedly according to the reaction conditions. This experimental finding is in contrast to the idea hitherto accepted that in the nucleophilic addition-type polymerization of α-amino acid $\text{N-}\text{carboxyanhydride}$ the growing chain end reacts preferentially with one of the enantiomorphic $\text{N-}\text{carboxyanhydrides}$ having the same configuration, and indicates the importance of the investigation of stereoselectivity in the $\text{N-}\text{carboxyanhydride}$ polymerization using suitable model reactions. Most $\text{(S)-α-}\text{amino}$ acid esters reacted preferentially with $\text{(R)-}\text{phenylalanine}\text{N-}\text{carboxyanhydride}$, and this type of stereoselectivity increased with the $\text{N-}\text{methylation}$ of $\text{N-}\text{carboxyanhydride}$ and with increasing bulkiness of the C^α substituent of α-amino acid esters (alanine < norleucine < leucine < valine). The relationship observed between the stereoselectivity and the structures of amines and $\text{N-}\text{carboxyanhydrides}$ was explained satisfactorily in terms of the transition state model in which the interaction of $\text{N-}\text{carboxyanhydride}$ nitrogen and α-amino acid ester carbonyl as well as the interaction of $\text{N-}\text{carboxyanhydride}$ carbonyl and α-amino acid ester nitrogen was taken into account. $\text{(S)-}\text{Proline}$ ethyl ester did not show enantiomer selectivity toward phenylalanine $\text{N-}\text{carboxyanhydride}$, but reacted preferentially with $\text{(S)-(N)-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$. for the reaction of proline ester with $\text{N-}\text{carboxyanhydride}$ a transition-state model was proposed, which was different from the transition state model proposed for other α-amino acid esters. Some experiments were carried out to examine the transition-state models proposed. The implications of the present investigation in stereoselectivity in the nucleophilic addition-type polymerization of $\text{N-}\text{carboxyanhydride}$ hitherto reported are discussed.     

Cytochrome oxidase from an extreme thermophile. Thermus thermophilus HB8

The cytochrome oxidase (EC 1.9.3.1) of $\text{Thermus}\text{thermophilus}$ HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme $\text{a}$ and heme $\text{c}$ as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with $\text{T}\text{.}\text{thermophilus}$ cytochrome c-552. The oxidation of $\text{T}\text{.}\text{thermophilus}$ cytochrome $\text{c}$-555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome $\text{c}$-552. The enzyme was highly stable to heat.     

Hydrogenase activity in the thermophile Mastigocladus laminosus

Hydrogenase activity in the thermophilic cyanobacterium, $\text{Mastigocladus}$$\text{laminosus}$ was studied both $\text{in vivo}$ and $\text{in vivo}$ hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H₂-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O₂-independent reaction. $\text{In vitro}$ hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for $\text{Anabaena cylindrica}$.     

The interaction between the heme c and heme d moieties of Pseudomonas nitrite reductase as revealed by magnetic and natural circular dichroism studies.

A possibility of a heme-heme interaction between the heme $\text{c}$ and heme $\text{d}$ moieties in $\text{Pseudomonas}$ nitrite reductase was examined by using magnetic and natural circular dichroism. The MCD of the heme $\text{c}$ moiety in the ferric enzyme was similar to that of mammalian ferricytochrome $\text{c}$ in shape and intensity, whereas in the reduced state the MCD intensity was considerably smaller than that of ferrocytochrome $\text{c}$. When the heme $\text{d}$ moiety was perturbed by the complex formation with CO, imidazole or cyanide as well as by pH changes, the depressed MCD was restored to the MCD level of mammalian ferrocytochrome $\text{c}$, accompanying conformational changes around the prosthetic groups. Thus, it was concluded that the heme-heme interaction exists only in the reduced enzyme and that this interaction is released under appropriate conditions.     

Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus.

In homogenates of $\text{Macaca}$$\text{mulatta}$ (Rhesus) or $\text{Cebus}$$\text{apella}$ amygdaloid nuclear complex, adenylate cyclase activity was approximately doubled by either 10μ$\text{M}$ dopamine or 8m$\text{M}$ NaF. In the presence of morphine, the stimulation by dopamine was reduced. A 90–100% inhibition of the dopamine stimulation was obtained with 20μ$\text{M}$, and a 50% inhibition, with 5μ$\text{M}$ morphine. The effects of 10μ$\text{M}$ morphine on dopamine stimulation were reversed by 10μ$\text{M}$ naloxone. Morphine itself did not significantly affect the basal adenylate cyclase activity, but in the presence of 10μ$\text{M}$ morphine the stimulation by 8m$\text{M}$ NaF was reduced approxiamtely 50%. The data suggest an action of morphine at a receptor site which is distinct from the dopamine receptor, but which inhibits the dopamine-stimulated adenylate cyclase. In addition, the cyclic GMP content of $\text{Cebus}$ amygdala slices was reduced by 50–75% during incubation for 5–20 minutes with morphine. Maximum effects on cyclic GMP were obtained with 10μ$\text{M}$, and half-maximum effects, with 0.1μ$\text{M}$ morphine. The effect of morphine on amygdala cyclic GMP was not reversed by naloxone. Thus, this action of morphine may not be receptor mediated, or may involve the interaction of morphine with receptors other than the opiate receptor.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

Effects of calcium and strontium on divalent ion content of refractive granules in Tetrahymena pyriformis

Quantitative electron probe analysis was employed to analyse refractile granules of T. pyriformis individually and in situ. The mean ratios of $\text{Ca}\text{P}$, $\text{Mg}\text{P}$, $\text{K}\text{P}$ and $\text{(}\text{Ca + Mg + K}\text{)}\text{P}$ in granules were similar in cells grown in three different nutrient media. Supplementing the calcium content of proteose-peptone medium with 0.3 and 3 mM calcium depressed the mean $\text{Ca}\text{P}$ ratio and increased the $\text{Mg}\text{P}$ ratio of the granules. The mean $\text{K}\text{P}$ ratio and $\text{(Ca + Mg)}\text{P}$ ratio were not significantly altered. When medium M was supplemented with 0.3 mM calcium, there was no significant change in the $\text{Ca}\text{P}$, $\text{Mg}\text{P}$, $\text{K}\text{P}$ nor $\text{(Ca + Mg)}\text{P}$ ratios. When supplemented with 3 mM Ca, the $\text{Ca}\text{P}$ ratio increased slightly, and the $\text{Mg}\text{P}$ ratio decreased slightly. There was no significant change in the $\text{K}\text{P}$ and $\text{(Ca + Mg)}\text{P}$ ratio. When each medium was supplemented with strontium, all granules incorporated this element, probably at the expense of calcium. The $\text{(Ca + Mg + Sr)}\text{P}$ ratios in granules in each strontium-containing medium were comparable to the $\text{(Ca + Mg)}\text{P}$ ratio in the granules in strontium-free media, indicating that the mix of divalent ions in granules may vary, but the proportion of divalent ions to phosphorus tends to be uniform.     

Site of action of the crinkled (cr) locus in the mouse.

The site of action of the crinkled (cr) locus was determined by combining dermis and epidermis from the tail of 15-day $\text{+}\text{cr}$ and $\text{cr}\text{cr}$ mice and by growing the recombined skins in the testes of histocompatible mice. Since $\text{cr}\text{cr}$ mice have bald tails, the presence or absence of hair in the graft was the feature used to determine gene action. Grafts of the combinations $\text{+}\text{cr}$$\text{epidermis}\text{-}\text{+}\text{cr}$ dermis and $\text{+}\text{cr}$$\text{epidermis}\text{-}\text{cr}\text{cr}$ dermis grew hair, whereas grafts of the combinations $\text{cr}\text{cr}\text{epidermis}\text{-}\text{+}\text{cr}$ dermis and $\text{cr}\text{cr}$$\text{epidermis}\text{-}\text{cr}\text{cr}$ dermis produced no hair. It was concluded that the cr locus, at least for tail skin, is active in the epidermis.     

DNA synthesis during the division cycle of three substrains of Escherichia coliBr

The relationship between chromosome replication and the bacterial division cycle has been examined in three substrains of Escherichia coli$\text{B}\text{r}$ obtained from different sources and designated $\text{B}\text{r}$ A, $\text{B}\text{r}$ F and $\text{B}\text{r}$ K. At growth rates greater than 1.0 doubling per hour (μ > 1.0), the time for a round of chromosome replication (C) was 42 minutes in all three substrains, but the time between the end of a round and cell division (D) was 22 minutes in $\text{B}\text{r}$ A, 16 minutes in $\text{B}\text{r}$ F and 14 minutes in $\text{B}\text{r}$ K. At slower growth rates C and D increased, but to significantly different extents in the three substrains. When μ = 0.5, C and D were approximately 80 and 40 minutes in $\text{B}\text{r}$ A, 60 and 20 minutes in $\text{B}\text{r}$ F, and 70 and 20 minutes in $\text{B}\text{r}$ K.As a consequence of the lengths of the C and D periods in the three stocks of E. coli$\text{B}\text{r}$, the patterns of chromosome replication during the division cycle differed. The most obvious difference was that E. coli$\text{B}\text{r}$ F and E. coli$\text{B}\text{r}$ K possessed periods devoid of DNA synthesis at both the beginning and the end of the division cycle during slow growth, whereas E. coli$\text{B}\text{r}$ A contained only one period devoid of DNA synthesis at the end of the cycle.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Capsaicin and potassium evoked substance P release from the nucleus tractus solitarius and spinal trigeminal nucleus invitro

The nucleus tractus solitarius and the spinal trigeminal nucleus receive peripheral sensory input from substance P containing afferent nerves. This study demonstrates that $\text{in}$$\text{vitro}$ depolarization of these nuclei in tissue slices evokes a calcium-dependent efflux of substance P immunoreactivity. Capsaicin (33μM) also elicits substance P release from the nucleus tractus solitarius and spinal trigeminal nucleus but not from the hypothalamus. The occurrence of potassium-stimulated SP release from the two medullary nuclei fulfills one of the criteria for neurotransmitter status. The capsaicin data support the contention that this agent elicits release of substance P from nuclear regions receiving peripheral afferent information in substance P nerves independent of the particular sensory modality served but is ineffective in nonsensory areas.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.