Studies on lysine analogs,aspartate-derived amino acids,and attempted mutant selection on oat seedlings

The lysine analogs S-2-aminoethyl-L-cysteine (AEC) and DL--hydroxylysine (DHL) caused severe growth inhibition in dark-grown oat seedlings (Avena sativa L. and A. nuda L.) at similar concentrations while L-lysine methyl ester (LME) had little effect. Lysine, arginine, and ornithine reversed the inhibition caused by AEC and DHL, the order of effectiveness being lysine>arginine>ornithine. Of aspartate-pathway amino acids, tested individually and in combinations for inhibitory effects on seedling growth, lysine and combinations containing lysine were the most inhibitory, but the inhibition was much less than that produced by AEC. Only slight synergistic effects occurred when oat seedlings were grown in the presence of paired combinations of aspartatepathway endproduct amino acids.Ca. 54,000 seeds obtained from 3,463 plants grown from ethyl-methanesulfonate (EMS) treated seed were screened for resistance to AEC. Three resistant variants were identified but the resistance was not recovered among their self-pollinated progeny.Abbreviations AEC S-2-aminoethyl-L-cysteine - DHL DL--hydroxylysine - EMS ethyl methanesulfonate - LME L-lysine methyl ester Paper No. 10351, Scientific Journal Series, Minnesota Agricultural Experiment Station     

Minimum structure opioids-dipeptide and tripeptide analogs of the enkephalins

Through a systematic reduction of peptide structure, a series of 25 tripeptide and 5 dipeptide amide and alcohol analogs of enkephalin were synthesized and assayed in vitro on the stimulated guinea pig ileum. Tyr-Pro-Phe-NH2, Tyr-D-Ala-Phe-NH2, Tyr-D-Ala-Phe-ol and Tyr-D-Phe-Phe-NH2 had 20-25% the potency of Met-enkephalin. Four aromatic alkylamides of the dipeptide Tyr-D-Ala were made with benzylamine, phenethylamine, phenylpropylamine and phenylbutylamine. All had full naloxone reversible enkephalin-like activity in the ileum assay. Tyr-D-Ala-phenylpropylamide has about 80% the potency of Met-enkephalin in vitro, and is equipotent with Tyr-D-Ala-Gly-Phe-Met-NH2 in producing analgesia in mice after intraventricular administration. Tyr-D-Phe-NH2 is the smallest peptide to show full intrinsic enkephalin-like activity in vitro, although its potency is very low.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala

The synaptology of neurotensin (NT)-, somatostatin (SS)- and vasoactive intestinal polypeptide (VIP)-immunoreactive neurons was studied in the central nucleus of the rat amygdala (CNA). Three types of axon terminals formed synaptic contacts with peptide-immunoreactive neurons in the CNA: Type A terminals containing many round or oval vesicles; Type B terminals containing many pleomorphic vesicles; and Type C terminals containing fewer, pleomorphic vesicles. Peptide-immunoreactive terminals were type A. All three types of terminals formed symmetrical axosomatic and asymmetrical axodendritic contacts. However, type B and peptide-immunoreactive terminals frequently formed symmetrical axodendritic synaptic contacts. VIP-immunoreactive terminals also formed asymmetrical axodendritic contacts. SS- and NT-immunoreactive terminals commonly formed symmetrical contacts on SS- and NT-immunoreactive cell bodies, respectively. VIP-immunoreactive axon terminals were postsynaptic to nonreactive terminals. Type B terminals appeared more frequently on VIP neurons than on NT or SS neurons.     

Somatostatin antagonist analog increases GH, insulin, and glucagon release in the rat

We have utilized the relative structural simplicity of several short, cyclic, highly active somatostatin analogs in the search for competitive antagonists of somatostatin. During an attempted synthesis of cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr), catalytic hydrogenation of the protected peptide intermediate unexpectedly gave cyclo [7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] in which the benzyl protecting group on Thr could not be removed even upon prolonged treatment under standard conditions. Injection of this new peptide into the rat completely blocked the inhibitory effects of exogenous somatostatin on GH, insulin, and glucagon release. Indeed, in fasted rats, basal hepatic portal insulin and glucagon levels were significantly increased after analog treatment. Plasma GH levels in Nembutal-anesthetized and stimulated rats were also increased after injection of the analog. These results provide strong evidence that endogenous somatostatin exerts local tonic control of pituitary and pancreatic secretions. The availability of a somatostatin anatagonist should be of considerable value in elucidating the roles of somatostatin in these and many other physiological processes.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Do neural crest cells in the pancreas differentiate into somatostatin-containing cells?

Summary The possibility that the somatostatin cells are derived from the neurectoderm has been questioned in avian embryos. Isotopic and isochronic transplantations of the neural primordium from quail into chick embryos were made at the vagal level (somites 1 to 7). Quail and chick cells can be distinguished by the structure of their nucleus. The somatostatin cells were characterized immunocytochemically. In no case did quail cells showing the immunological reaction originate from the neural crest.     

Interaction of high-affinity nucleotide binding sites in mitochondrial ATP synthesis and hydrolysis

The present study contributes to the problem of the dynamic structure of mitochondrial F1-ATPase and the functional interrelation of so-called tight nucleotide binding sites. Nucleotide analogs are used as a tool to differentiate two distinct functional states of the membrane-bound enzyme, proposed to reflect corresponding conformational states; they reveal F1-ATPase as a dual-state enzyme: ATP-synthetase, and ATP-hydrolase. The analogs used are 3-naphthoyl esters of AD(T)P, and 2(3)-O-trinitrophenyl ethers of AD(T)P. Both types of analogs act inversely to each other with respect to their relative effects on oxidative phosphorylation and on ATPase in submitochondrial vesicles. The respective ratios ofK _i versus both processes are 250/1 compared to 1/170. It is also shown that in the presence of the inhibitory 3-esters oxidative phosphorylation deviates from linear kinetics and that these inhibitors induce a lag time of oxidative phosphorylation depending on the initial pattern of nucleotides available to energized submitochondrial vesicles. The duration of the lag time coincides with the time course of displacement of the analog from a tight binding site. The conclusions of the study are: (a) the catalytic sites of F₁-ATP-synthetase are not operating independently from each other; they rather interact in a cooperative manner; (b) F₁-ATPase as a dual-state enzyme exhibits highly selective responses to tight binding of nucleotides or analogs in its energized (membrane-bound) state versus its nonenergized state, respectively.Abbreviations used: N-AD(T)P, 3-O-naphthoyl(1)-AD(T)P; DMAN-AD(T)P, 3-O-(5-dimethylaminonaphthoyl(1))-AD(T)P, also termed F-AD(T)P in previous papers because of its fluorescence; TNP-AD(T)P, 2(3)-O-(2,4,6-trinitrophenyl)-AD(T)P; FCCP,p-trifluoromethoxycarbonylcyanide phenylhydrazone.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.