Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle

Summary We have compared some features of the resting and the insulin-stimulated uptake of -aminoisobutyrate (AIB) in frog skeletal muscle. We found a substantial difference between the two processes, namely, that resting AIB uptake is Na-independent while the insulin-stimulated fraction of the AIB uptake is Na-dependent.Since the amino acid transport systems in frog skeletal muscle are poorly characterized, we have also surveyed some of their properties. One of the most interesting findings of this survey is that both the uptake and efflux of AIB are inhibited by low concentrations of PCMBS (parachloro-mercury-benzene sulfonic acid 5×10^–5 m). In contrast, the carrier mediated transport of basic amino acids is neither inhibited by this mercurial agent nor accelerated by insulin.The action of PCMBS strongly suggests the presence of a critical sulfhydryl group in the amino acid carrier system utilized by AIB. This group is exposed to the outside solution since PCMBS penetrates cell membranes poorly, and in addition its inhibitory actions were reverted by agents that do not penetrate the cell membrane like albumin or glutathione.     

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.     

Leu-enkephalin-stimulated growth hormone and prolactin release in the rat: comparison with the effect of morphine

The effect of Leu⁵-enkephalin on growth hormone (GH) and prolactin (PRL) release was studied $\text{in vivo}$ in the infant rat and compared to that of morphine. In 10 day-old pups, intracerebroventricular injection of Leu⁵-enkephalin (50, 75 and 100 μg) resulted in a dose-related increase in plasma GH; morphine was active as GH releaser at the dose of 5 and 10 μg, but not at 2.5 μg. Pretreatment with naloxone (2 mg/kg ip) suppressed the GH-releasing effect of either Leu⁵-enkephalin (100 μg) or morphine (10 μg). Leu⁵-enkephalin (75 and 100 μg) induced a rise in plasma PRL which was neither dose-related nor antagonized by naloxone; morphine (5 and 10 μg) was active as PRL releaser and its effect was antagonized by naloxone. These results indicate that: 1) Leu⁵-enkephalin stimulates both GH and PRL release; 2) the release of GH by Leu⁵-enkephalin but likely not that of PRL involves specific opiate receptors; 3) morphine releases GH and PRL through specific opiate receptors.     

Preferred conformations of protected homo-oligo-L-glutamate peptides in CDCl3 and CDCl3/TFA mixtures

A conformational analysis of protected glutamate homo-oligopeptides Z-[Glu(OEt)]_n-OEt (n = 2–7) was carried out in chloroform solution using high-resolution ¹H-nmr spectroscopy. At dilute peptide concentrations, the backbone NH and α-CH resonances are well resolved and can be assigned by combining extensive homonuclear decoupling experiments with data for co-oligopeptide derivatives. The structure of these peptides in solution was then assessed using information from chemical shifts, coupling constants, temperature coefficients, and titration of each oligomer with trifluoroacetic acid (TFA). The di- and tripeptides are found to be in disordered forms in deuterochloroform (CDCl₃) and CDCl₃/TFA mixtures. The tetrapeptide exhibits a folded structure with intramolecular hydrogen bonding at Glu² in CDCl₃ and undergoes a transition to increasingly disordered forms as TFA is added. The pentamer to heptamer show a folded structure with a strong intramolecular hydrogen bond at Glu² and a weaker hydrogen bond at Glu³, which are disrupted as these peptides go to random coils at high TFA/CDCl₃ ratios. In addition, the N-terminal portions of these glutamate peptides appear to be involved in side chain–main chain interactions. The results support the hypothesis that protected linear homo-oligopeptides may possess two or more segments of conformation with intramolecular folding preferred near the N-terminal portion.     

Intralymphatic administration of BCG in melanoma patients

Summary From November 1973 to December 1974, 20 patients with advanced malignant melanoma were treated with BCG given by intralymphatic route at the Cancer Institute of Milan. The lyophilized Pasteur BCG was used. Patients were treated with a single dose ranging from 0.2–80 mg. Patients' performance status was never severely impaired.The most frequent side effects were fever, lymphangitis, and lymph node enlargement.Variations were observed in white cell count, ERS and immunoglobulins; in no case did we find evidence of liver toxicity or tumor growth enhancement. It is concluded that the intralymphatic route is a safe way of administrating BCG.     

1H-nmr studies of polyoxyethylene-bound homo-oligo-L-methionines

The use of ¹H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz ¹H-nmr study of POE-bound homo-oligo-L -methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl₃ are qualitatively similar to those of the low-molecular-weight Boc-L -Met_n-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L -Met)_n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L -Met)_n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6–7 Hz ³J_NH-CH coupling constants.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.     

Amblyomma americanum: characterization of salivary prostaglandins E2 and F2 alpha by RP-HPLC/bioassay and gas chromatography-mass spectrometry.

Secretagogue-induced saliva of the tick, Amblyomma americanum (L.) was fractionated by reversed-phase-high-performance liquid chromatography (RP-HPLC) and bioassayed in smooth muscle preparations. Material with retention times of authentic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) were found to cause contraction of preparations of rat colon and rat stomach strips. Gas chromatography-mass spectra of selected ions of both HPLC-purified fractions confirmed the existence of PGE2 and PGF2 alpha. Bioassay of individual samples obtained from ticks stimulated to salivate with pilocarpine, dopamine + theophiline, or dopamine + theophiline + GABA indicated that all these secretagogues induced similar amounts of prostaglandin secretion, averaging 469 ng PGE2/ml. These pharmacological doses of prostaglandin are hypothesized to assist in tick feeding by inducing vasodilation and/or other pharmacological events in their hosts.