Effect of Pro-Leu-Gly-NH2 on plasma levels of α-MSH in the rat

With the idea to give further support that Pro-Leu-Gly-NH₂ (PLG) acts as MIF (the inhibitor factor of MSH), this paper reports the effect of PLG on the secretion of MSH release using a recently developed radioimmunoassay for α-MSH.Pro-Leu-Gly-NH₂ was effective in inhibiting the release of MSH induced by the injection of acid extracts of median eminence (MRF). The rise in plasma MSH by these extracts was not due to their intrinsic content of MSH. Pro-Leu-Gly-NH₂ also inhibited the basal secretion of MSH in male rats. Since PLG also blocked the release of MSH induced by the injection of haloperidol, it is suggested that its effect is not mediated by dopamine.     

Hydrogen bonds in the tripeptide Pro-Leu-Gly-NH217O and 1H studies

¹⁷ONMR measurements of labeled Pro-Leu-Gly-NH₂ were carried out at different pH levels and in mixed solvents of water/acetonitrile. Complementary studies of the amide protons were carried out in acetonitrile-d₃. Only the prolyl $\text{C}\text{=}{}^{17}\text{O}$ group was sensitive to the pH level. Protonation of the amine group resulted in an upfield chemical shift of 18 ppm. The chemical shifts of each of the three oxygen sites was sensitive to the ratio water: acetonitrile. Solvent composition dependence of the chemical shift and linewidth suggests that the prolyl $\text{C}\text{=}{}^{17}\text{O}$ is involved in intramolecular hydrogen bond formation when Pro-Leu-Gly-NH₂ is dissolved in acetonitrile, while in water there is no intramolecular H bond.     

High-performance liquid chromatographic separation of ascorbic acid,erythorbic acid,dehydroascorbic acid,dehydroerythorbic acid,diketogulonic acid,and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH₂ analytical column, with acetonitrile: 0.05 m KH₂PO₄ (75:25, $\text{w}\text{w}$) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.     

Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines

Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase $\text{in}$$\text{vitro}$. Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the $\text{in}$$\text{vivo}$ effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

2-fluorourocanic acid, a potent reversible inhibitor of urocanase.

The K_i for the interaction of 2-fluorourocanic acid with urocanase (from $\text{Pseudomonas fluorescens}$) is 1000 times as great as K_m for the natural substrate, urocanic acid, whereas enzymatic hydration of the fluoro analog occurs $\text{ca}$. 100 times more slowly. Inhibition is competive and is eventually overcome by utilization of the analog. By contrast, 4-fluoro- and 2-amino-urocanic acid are neither significant inhibitors nor substrates for the enzyme. 2-Fluorourocanic acid may prove a useful tool for blocking the utilization of histidine as a one-carbon source in metabolism.     

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells.

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O₂ oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from ¹⁴C/³H leucine studies suggest that MSH may cause increased $\text{de}$$\text{novo}$ synthesis of tyrosinase.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

Utilization of C20-polyunsaturated fatty acids by a yeast fatty acid desaturase mutant

A study was made of the utilization of C₂₀-polyunsaturated fatty acids by the $\text{S. cerevisiae}$ fatty acid desaturase mutant $\text{olel-1}$, Arachidonic acid, 8,11,14-eicosatrienoic acid, and 5,8,11,14,17-eicosapentaenoic acid were about equally effective in supporting growth with lactate as the carbon source. The relative proportion of these fatty acids in total cell fatty acids was $\text{ca}$. 50%. 5,8,11-eicosatrienoic acid synthesized from oleate was less effective. Very little growth occurred with 11,14,17-eicosatrienoic acid or with 11,14-eicosadienoic acid. These results indicate the usefulness of the yeast mutant as a eucaryotic model for study of membrane systems enriched in specific C₂₀-polyunsaturated fatty acids.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Lack of growth hormone-releasing activity of (pyro)Glu-Ser-Gly-NH2

Youdaev $\text{et al.}$ (1) reported that (pyro)Glu-Ser-Gly-NH₂ isolated from bovine hypothalami or made synthetically stimulates the release of growth hormone (GH). Therefore, we synthesized the tripeptide and tested it in several $\text{in vivo}$ and $\text{in vitro}$ assay systems for GH releasing activity. Our results demonstrate that (pyro)Glu-Ser-Gly-NH₂ does not stimulate the release of immunoreactive GH from rat pituitaries $\text{in vitro}$ in doses of 0.1–1000 nanog/ml. Similarly, (pyro)Glu-Ser-Gly-NH₂, injected intravenously into rats in doses of 1μg and 10μg/rat or infused into a hypophysial portal vessel in doses of 0.01 μg and 0.1 μg/rat did not increase serum GH levels as measured by radioimmunoassay. When this tripeptide was injected intravenously in doses of 500 μg into sheep it did not raise plasma GH levels. These results demonstrate that under the conditions of this investigation (pyro)Glu-Ser-Gly-NH₂ does not display any GH-releasing activity.     

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.     

Equilibrium centrifugation of proteins in acid solutions. Ovalbumin in aqueous acetic, propionic, and butyric acids.

The use of short-chain aliphatic acids in conjunction with the partial specific volume of the native protein, v?_p for molecular weight estimations and for the study of macromolecular interactions was further tested: Equilibrium centrifugation of ovalbumin has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. Under these experimental conditions there are two potential sources of error associated with the use of v?_p. These are: (a) preferential interaction with solvent components[$\text{Γ = (}\text{δm}{}_3\text{δm}{}_2\text{)μ}{}_1\text{,μ}{}_3\text{T, P}$] and (b) volume changes accompanying the exposure of the protein to acid (δv?_p). Volume changes associated with the transfer of ovalbumin to acid solutions have been measured. With this information at hand, it was possible to show that δv?_p and Γ in these solvent systems are both small; therefore, v?_p can be used instead. This work has also shown that the behavior of ovalbumin is distinct from that of β-lactoglobulin and aldolase. Thus, it appears that each protein has a characteristic behavior in these aqueous organic solvents which is related to some aspect of protein structure. Ovalbumin undergoes a series of reversible and irreversible interactions in these solvent systems which are dependent on both the length of the acid's carbon chain and the concentration of acid. An effort has been made to elucidate these interactions in terms of simple models.     

Basal and potassium stimulated, calcium dependent, endorphins release from pituitary cells.

Mouse pituitary tumor cells grown in tissue culture release endorphins spontaneously to the culture medium. Depolarization of these cells by incubation with high K⁺ concentration (56 mM) increased 2–3 folds the release of endorphins. The K⁺ evoked release was Ca⁺⁺ dependent by that: $\text{a}$, removal of Ca⁺⁺ ions inhibited 90% of K⁺ stimulated release. $\text{b}$, ethyleneglycol-bis (β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) inhibited release of endorphins in the presence of high K⁺ and Ca⁺⁺. It is suggested that dual regulatory system inhibit and/or stimulate $\text{in-vivo}$ release of endorphins from the pituitary glands.     

Effect of bombesin-stimulated gastrin on gastric acid secretion in man

The effect of bombesin on gastrin release and gastric acid secretion was investigated in 10 healthy volunteers. Bombesin (0.6 μg · Kg^?1 · hr^?1) produced a significantly higher $\text{(}\text{p}\text{<}\text{0.001)}$ increase in plasma gastrin levels (86.7 11.1 pmo/1 than after a protein meal (39.6 ± 5.6 pmol1/1). The gastric acid secretory response to bombesin (12.1 ± 2.9 mEq · hr^?1) was however significantly lower $\text{(}\text{p}\text{<}\text{0.005)}$ than the maximal response produced by pentagostrin (20.9 ± 3.5 mEq · hr^?1) at the dose of 6 μg · Kg^?1. Atropine did not modify gastrin release induced by bombesin but significantly reduced gastric acid secretion $\text{(}\text{p}\text{<}\text{0.01)}$. From the data presented it may be hypothesized that less biologically active forms of gastrin and/or other peptides inhibiting the gastrin effect upon gastric acid secretion may be released by bombesin.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

The amino terminal sequences of acid proteases-human pepsin and gastricsin and the protease of Rhizopus chinensis.

The amino acid sequences near the amino termini of human pepsin (34 residues) and gastricsin (24 residues) and the acid protease from $\text{Rhizopus chinensis}$ (27 residues) have been determined using automated Edman degradation. From these results three additional observations were made. First, two structural variants have been observed for human gastricsin and for the $\text{Rhizopus}$ protease. Both cases are apparently genetic in origin. Second, a stretch of sequence in the $\text{Rhizopus}$ protease, residues 14 to 26, is highly homologous to the known sequence of porcine pepsin at the region of residues 11 to 23. Third, the sequences of the NH₂-terminal region of human pepsin and gastrisin are homologous.     

Inhibition of porphobilinogen synthase by heme and an enol-ether amino acid

The enol-ether amino acid, L-2-amino-4-methoxy-$\text{trans}$-butenoic acid (AMTB) is an inhibitor of porphobilinogen synthase (PBG synthase) when added prior to the addition of the substrate δ-aminolevulinic acid. The inhibition of PBG synthase by several stereoisomers and analogues of AMTB was investigated to determine those structural features of AMTB which may be necessary for inhibition. The D-$\text{trans}$ isomer was also an inhibitor after preincubation, whereas the L-$\text{cis}$ isomer inhibited with or without preincubation. The amino acid analogues, DL-vinylglycine, DL-2-aminobutanoic acid, the reduced form of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid, L-2-amino-4-(2-aminoethoxy)-$\text{trans}$-3-butenoic acid and its reduced congener did not inhibit PBG synthase even with preincubation. This structure activity relationship indicates that the $\text{trans}$ double bond and methoxy moiety of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid are probably required for inhibition.Heme, when preincubated with PBG synthase, was an inactivator of the enzyme. However, when both L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid and heme were simulatneously preincubated with PBG synthase, inactivation of the enzyme was greater than with either compound separately. The possibility of multiple catalytic sites was suggested by the use of multiple inhibition kinetics in the presence of heme and L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.