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Activation of delta-opioid receptors (DOR) attenuates anoxic K(+) leakage and protects cortical neurons from anoxic insults by inhibiting Na(+) influx. It is unknown, however, which pathway(s) that mediates the Na(+) influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na(+) influxes mediated by voltage-gated Na(+) channels; (2) DOR activation inhibited Na(+) influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K(+) derangement; and (3) DOR activation had little effect on Na(+)/Ca(2+) exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K(+) derangement by restricting Na(+) influx mediated by Na(+) channels and NMDA receptors, and that non-NMDA receptors and Na(+)/Ca(2+) exchangers, although involved in anoxic K(+) derangement in certain degrees, are less likely the targets of DOR signal.

The N-terminal dipeptide Tyr-d-Ala of a mu-selective agonist, dermorphin tetrapeptide (DT, H-Tyr-D-Ala-Phe-Gly-NH2) and delta-selective agonist deltorphin C (DEL-C, H-Tyr-D-Ala-Phe-Asp-Val-Val- Gly-NH2) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2-bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra- and heptapeptides I-VI were characterized for mu- and delta-opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N-terminal depeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to mu- and delta-prototypes (500-fold on the mu-site, analog I, and 125-fold on the delta-site, analog IV). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA2 values of 6.05 and 6.82. Analog IV did not exhibit the delta-antagonist potency and delta-selectivity of TIPP peptides.

A new class of δ opioid antagonists was recently discovered in which the sequence Tyr-Tic was used as a message domain. The substitution of Tyr1 by Dmt enhanced the δ selectivity and antagonist activity. The excellent properties of these ligands stimulated us to prepare the corresponding tritiated derivatives. Peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, with a reduction in opioid activity. To avoid this side-reaction we synthesised the N,N-dimethylated analogue (N,N(Me)2-Dmt-Tic-OH), which was found to be stable. On the basis of this result, we prepared diiodinated analogues of H-Dmt-Tic-OH and N,N(Me)2-Dmt-Tic-OH to undergo catalytic dehalotritiation. Products of high specific radioactivity were obtained: 44.67 Ci/mmol for [3H]Dmt-Tic-OH and 59.88 Ci/mmol for [3H]N,N(Me)2-Dmt-Tic-OH. Copyright © 1998 John Wiley & Sons, Ltd.

A series of deltorphin C (H-Tyr- d -Ala-Phe-Asp-Val-Val-Gly-NH 2 ) analogues were synthesized to assess the consequences of changing anionic and hydrophobic residues on δ receptor selectivity. Analogues with altered C-terminal groups, inverted sequences, or esterified with tert -butyl, benzyl, or ethyl groups revealed that high δ selectivity required an unmodified amino acid sequence. Shifts of Asp and hydrophobic residues decreased δ selectivity due to loss in δ affinity (5- to ≈ 700-fold); μ affinity was unchanged or increased 14-fold. Suppression of charge or deamidation diminished δ selectivity through reduced δ and modified μ affinities. Data provide evidence that a negative charge does not a priori guarantee high selectivity and specific alignment of anionic and hydrophobic residues might facilitate optimum spatial configuration which complements the δ receptor binding site.

BACKGROUND: Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). MATERIALS AND METHODS: Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. RESULTS: H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. CONCLUSIONS: Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.

The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.

Substitution of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in place of Gly2 in dynorphin A-(1-13)-NH2 and -(1-11)-NH2 (DYN) analogues (1 and 2) decreased the affinity to the kappa, delta, and mu receptors, and kappa selectivity. The analogue [D-Ala2, des-Gly3]DYN (4), a chimera between deltorphin/dermorphin N-terminal tripeptide and DYN, was virtually inactive for kappa-sites while the affinities for delta- and mu-receptors remained essentially unchanged. The doubly substituted analogue [2',6'-dimethyl-L-tyrosine (Dmt1)-Tic2]DYN (3) exhibited high delta-affinity (Ki=0.39 nM) while mu- and kappa-affinities were only an order of magnitude less (4-5 nM). Bioactivity of [Tic2]DYN peptides (1-3) on guinea-pig ileum and rabbit jejunum revealed potent delta- and kappa-antagonism, while the delta agonist potency of 4 was comparable to DYN. Thus, conversion from a kappa-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with delta- and mu-opioid agonists containing a Tic2 residue.

We have recently designed potent delta selective opioid antagonist dipeptides on the basis of a simple conformational analysis. Following a similar procedure we found a mu selective dipeptide antagonist, 2,6-dimethyl-Tyr-D-Phe-NH2. Although its selectivity is not as high as those of the quoted delta selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6-dimethyl-Tyr-D-Phe message, like the delta selective 2,6-dimethyl-Tyr-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid counterpart, seems able to impart antagonism to longer peptides.

The surprising change of selectivity induced by the change of chirality in peptides containing the tetrahydro-3-isoquinoline carboxylic acid (Tic) in second position, interpreted as a conformational preference induced on the Tyr-Xaa-Phe domain, can instead be attributed to the Tyr-Tic message domain. The relative spatial disposition of the aromatic rings of δ-selective non peptidic opiates is compatible with a message domain, in opioid peptides, of only two residues. This hypothesis was tested through the synthesis of Tyr-L-Tic-NH 2 , Tyr-D-Tic-NH 2 , Tyr-L-Tic-Ala-NH 2 , Tyr-L-Tic-Ala-OH and Tyr-D-Tic-Ala-NH 2 . Peptides containing Tyr-L-Tic- behave as very selective δ antagonists and those containing Tyr-DTic- as non selective agonists. This is the first case of opioid peptides containing a two-residue message domain and of opioid dipeptides with substantial opioid activity.

Sixteen dermorphin analogues were synthesized and characterized for mu- and delta-opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or the C-terminal residue; deletions of Gly4 or Pro6-Ser7; inclusion of Z or an acetyl group on the epsilon-amino group of Lys7; and the presence of either a C-terminal amide or free acid group. Two peptides, [Lys7-OH]- and [Lys7-NH2]dermorphin, had mu-affinities (Ki mu = 0.15-0.13 nM) and mu-selectivities (Ki delta/Ki mu = 1158-1482) higher than dermorphin (Ki mu = 0.28 nM; Ki delta/Ki mu = 295) and best fitted a one-site binding model similar to dermorphin. Significantly better (P < 0.0001) fits to a two-site binding model vs. a one-site model were observed with four dermorphin analogues: [Lys(Z)7-OH]heptapeptide, [des-Gly4(Tyr4,Pro5,Asn6-OH)]hexapeptide and two pentapeptides, [Tyr5-NH2] and [Trp4,Asn5-OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain mu-receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of mu-receptors; however, only detailed analyses of the data derived from the non-linear regression fits for one- or two-components gave evidence for the possible existence of two separate [3H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two-site model (P < 0.05), but only four seemed to have two distinct Ki values (P < 0.0001).

Analysis of deltorphin A position 4 analogues included: backbone constrained N α MeHis, spinacine (Spi), N α MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His4 enantiomers. Highδ selectivity was lost with the following replacements: N α MeHis4, N α MePhe4 and Phg4 reducedδ binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi4 or Tic4 increasedµ affinity. Imidazole methylation of His4 marginally affected opioid binding and doubledδ selectivity; alkylatedd-His4-derivatives generally maintainedδ selectivity in spite of decreasedδ affinities. Thus, His4 imidazole preservesδ selectivity by facilitating highδ binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His4 imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr1 suggestive that this conformation might contribute to highδ affinity and selectivity.

The peptidase-resistance of deltorphins (DEL-A, H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and (DEL-C, H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), highly selective and potent agonists of the delta opioid receptor, were investigated in vitro. DEL-C was fully resistant to degradation by rat plasma and strongly resistant to degradation by rat brain homogenates. DEL-A was cleaved with a half-life of 131.6 min upon incubation with plasma, and 57.4 min with rat brain homogenates. N-terminal truncated sequences of DEL-A and -C with free carboxyl groups, were also analyzed for receptor binding activity using [3H] DADLE for delta sites and [3H] DAGO for mu sites. The high enzymatic stability associated with deltorphins and their degradation products may make them prime candidates to characterize the role of delta-receptors in vivo.

Deltorphin N-terminal tetrapeptides [DEL A: H-Tyr-D-Met-Phe-His-R, where R = -NH2, -NH-NH2, -OCH3, -OH, -NH-NH-CO-R' (R' = -CH3 or adamantane); DEL C: H-Tyr-D-Ala-Asp-R (R = -OH, -NHCH3)], were used in a receptor binding assay with [3H]DADLE and [3H]DPDPE for delta sites, and [3H]DAGO for mu sites; tetrapeptide Ki delta values were similar with either [3H]-delta ligand. DEL A tetrapeptides C-terminally substituted with -NH2, -NH-NH2, -OCH3, and -OH had 10 to greater than 1,000-fold decreased Ki delta values, while Ki mu increased 5 to 100-fold to yield mu selectivity. C-Terminal substitution with -NH-NH2 and -OCH3 conferred highest mu selectivities; adamantyl and acetyl hydrazide derivatives were non-selective. DEL-(1-4)-OH peptides had decreased delta and mu affinities: DEL A-[Asp4]-(1-4)-OH and DEL C-(1-4)-OH had low affinities (greater than 1 microM), however, the Ki delta of the former was 5-fold greater than the latter, and the Ki mu was less by 15-fold. The data suggest that the "message" domain of DEL exhibits receptor selectivity different from that of the heptapeptide.

Deltorphins A, B, and C exhibit high delta-affinities (Ki = 0.12-0.31 nM) and very high delta-receptor binding selectivities (Ki mu/Ki delta = 1800-4100). A study of the delta-receptor binding properties of 15 deltorphin analogues focused primarily on the influence of the anionic group in the C-terminal tetrapeptide. Amidation of the beta-carboxyl groups of [Asp7], [Glu4], or [Asp4] in deltorphins A, B, and C, respectively, yielded peptides with enhanced mu-receptor affinities and minor changes in delta-affinities (Ki delta = 0.20-0.65 nM), but 5-8-fold diminished delta-selectivities. A free C-terminal carboxyl group markedly reduced delta-affinities and decreased delta-selectivities 6-11-fold; thus, the C-terminal amide group critically facilitates delta-affinity. Modifications in the anionic charged group or hydrophobic residues in the C-terminal tetrapeptide address domain of deltorphin A altered spatial distributions critical for delta-affinity and selectivity; e.g., [Nle6]deltorphin A enhanced mu-affinity and lowered delta-selectivity by two-thirds; the progressive, step-wise repositioning of Asp in deltorphin C (from position 4 to 7) was accompanied by linear decreases in delta-affinities and -selectivities, and increased mu-affinities of these peptides; enhancement of the charge density to -2, in [Asp6, Asp-OH7]deltorphin A, decreased delta-affinity and -selectivity, while [Asp4,5,His7]deltorphin A bound to neither mu- nor delta-sites. These results demonstrate that while an anionic group may occasionally facilitate high delta-receptor affinity, it represents an absolute requirement for the high delta-binding selectivity of these peptides. The locations of the charged groups relative to hydrophobic residues in the address domain of the peptide are also critical determinants for both delta-affinity and -selectivity.

Summary The replication of simian virus 40 (SV40) DNA is arrested by superinfection with frog virus 3 (FV3). Most of the SV40 DNA molecules synthesized before infection by FV3 become nicked or fragmented. These events take place at 37 °C, a restrictive temperature for FV3 growth.

The association of the replicase of foot-and-mouth disease virus (FMDV) with cellular membranes was suggested following attempts to produce a soluble enzyme by the use of detergents (Arlinghaus & Polatnick, 1967, 1969; Delagneau, 1971), as described for the purification of poliovirus replicase (Ehrenfeld, Maizel & Summers, 1970). Subsequently, the association with cellular membranes of the replicase and the single-stranded and replicative forms of RNA was reported for poliovirus (Caliguiri & Tamm, 1969, 1970a, b), for Semliki Forest virus (Friedman et al. 1972) and for tobacco mosaic virus (Ralph, Bullivant & Wojcik, 1971). In each case, the replicase-membrane complex contained endogenous RNA template (Arlinghaus & Polatnick, 1969; Caliguiri & Tamm, 1969; Ehrenfeld et al. 1970; Friedman et al. 1972). We show in this communication that FMDV replicase is associated with ribosome-containing membranes and requires glycerol to stabilize its activity during sedimentation in the presence of detergents.

Summary A cell-free system synthesizing FMDV RNA has been shown to be inhibited efficiently by purified FV3 particles. Neither vaccinia nor herpes virus particles produced this effect. The results obtained provide direct evidence that a structural component (or components) or FV3 is responsible for the inhibition.

A simple and unique negative viscosity —positive density gradient has been devised which enables an effective single step purification of foot-and-mouth disease virus (FMDV) directly from cytoplasmic lysates.

Skin of the frog Phyllomedusa sauvagei contains a cDNA sequence that codes for the selective mu-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide (DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for delta-type opioid receptors in rat brain synaptosomes. The IC50 values for DGAP on mu- and delta-receptors were only 28 microM and 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nM for delta-receptors and greater than 1 microM for mu-receptors yielding a very high delta selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for delta-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2 residue and is a selective mu-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem. 264, 354-362), exhibits a SR of 0.0055 similar to that for the conventional mu-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for mu- and delta-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.

Although the PHLIPs are able to enhance gastrointestinal contractility to a very modest extent, the actual physiological significance of the PHLIPs is not completely understood at the present time. Should the high Mr PHLIM be the precursor molecule(s), the PHLIPs may represent partial enzymatic hydrolysis products of the amino-terminal region of the precursor(s). In this regard, it is interesting to note that immunohistochemical staining of rat duodenum indicates the presence of PHY antiserum cross-reacting material in Brunner's glands, which appear to be involved in secretion of mucin. The amphibian peptide, bombesin, has also been detected in amphibian skin mucous glands by immunochemistry; other investigators have indicated that cerulein, an amphibian analog of the carboxy-terminal region of cholecystokinin, is discharged along with mucin from cutaneous glands in Xenopus after the injection of adrenalin.

A comparison of the effect of bombesin, dermorphin and salmon calcitonin on gastric acid secretion was assayed in rats two hours after their intracerebroventricular or subcutaneous administration. All three peptides significantly reduced gastric acid output after central administration. The order of potency was: bombesin greater than salmon calcitonin greater than dermorphin. After subcutaneous injection salmon calcitonin was very potent in reducing gastric acid output, while dermorphin was active only at high doses; bombesin had a very weak effect on the same parameter. Time-course studies demonstrated that the effect of these peptides reached a peak 1-2 h after their central administration, followed by a slow recovery toward control values. Salmon calcitonin, however, has a very prolonged action on the gastric parameters studied. These data indicate that bombesin and dermorphin might act centrally to modulate gastric acid secretion in the rat, while the very potent intracerebroventricular and subcutaneous action of salmon calcitonin suggests a different mechanism of action.

The effect of centrally administered dermorphin and a series of dimeric dermorphin analogs on gastric acid secretion was studied in pylorusligated rats. Dimeric dermorphin analogs consist of identical peptide chains either directly linked with a dihydride bond or a polyethyleneamine bridge at their carboxy termini. At a dose of 0.5 nmol dermorphin, and the dimeric analogs di-[D-Arg2,Sar4]-tetra-DM2, di-penta-DM0, di-[Sar4]-penta-DM0, di-[D-Arg2,Sar4]-penta-DM0, di-[D-Ala4]-penta-DM0 and di-DM0, and di-DM0, inhibited gastric acid output in a statistically significant manner (P less than 0.05). Furthermore, the binding characteristics of these peptides for the mu-type opioid receptor were analyzed using a brain synaptosomal fraction. Dermorphin and several dimeric dermorphin peptides bound to the mu-receptor: di-penta-DM0, di-[Sar4]-penta-DM0, di-penta-DM2 and di-DM0, had 3-to 5-fold greater affinity for the mu-receptor than the specific mu-agonist DAGO. These data indicate that a correlation exists between the central mediated gastric inhibitory effect of DM and several dimeric analogs and their affinity for the mu-type opioid receptor in rat brain.

The concept of using adenine nucleotide derivatives as general ligands in affinity chromatography was first proposed by Mosbach and his coworkers (1-3). Various immobilized adenine nucleotide derivatives have been prepared and used in the selective purification of enzymes by means of general ligand affinity chromatography (11, 17–21). From the efforts of many workers it is apparent that further development of this methodology would greatly accelerate our understanding of enzymes and their utilization in biochemical research, clinical chemistry and industrial production. Selective purification of enzymes by general ligand affinity chromatography is based on the principle that a single immobilized general ligand has the capacity to adsorb a large number of enzymes, or a given family of enzymes, which either require the same cofactor or have a common inhibitor or activator.

The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.

The metastatic human lung tumor, small cell carcinoma (SCC), exhibits ectopic (inappropriate) synthesis of numerous peptide hormones, such as ACTH, [Arg8]-vasopressin, hCGβ, calcitonin, oxytocin, prolactin, and growth hormone (Rees et al. 1974; Rees 1976; Baylin and Mendelsohn 1980). Furthermore, tumors resected from patients (Wood et al. 1981), or passaged in nude (athymic) mice (Erisman et al. 1982), or cultured into numerous cell lines (Moody et al. 1981, 1983 a, b; Sorenson et al. 1982) were found to contain immunoreactivity to the amphibian peptide bombesin and more recently to physalaemin (Erisman et al. 1982; Lazarus et al. 1983), which is the prototype of the tachykinin family of peptides discovered by Erspamer et al. (1964). The presence of immunoreactivity to bombesin was detected in human fetal and neonatal lungs (Wharton et al. 1978) and in the lungs of children (Cutz et al. 1981; Track and Cutz 1982) and appears to be associated with a mammalian form of bombesin similar to porcine gastrin-releasing peptide, a 27 amino acid residue peptide (Iwanago 1983; Price et al. 1983; Tsutsumi et al. 1983). On the other hand, the physalaemin-like immunoreactive material (PLIM), initially discovered in the gastrointestinal tract (Lazarus and DiAugustine 1980; Lazarus et al. 1980), was also present in neuronal (Lazarus et al. 1980) and pulmonary tissue from diverse mammlian species (Lazarus et al. 1982). Further studies showed however, that PLIM differed from the amphibian peptide (Lazarus et al. 1982) and the purified immunoreactive material was in fact an octapeptide with homology to the N-terminal region of physalaemin (Wilson et al. 1984).

The calcium antagonist, nifedipine (Adalat; Bayer), has previously been thought by most workers to lack cardiac-anti-arrhythmic properties, thus differing from verapamil. We have found that nifedipine was successful in converting cardiac glycoside (K-strophanthoside)-induced ventricular tachycardia to sinus rhythm in 4 of 5 anaesthetized dogs when given intravenously. In this respect, therefore, nifedipine is similar to verapamil. Further work is advocated before applying these results to the clinical situation in man.

The carboxyl terminal region of corticotropin (ACTH), (Ala34-Phe-Pro-Glu-Leu-Phe39), a region of the hormone conserved during evolution, served as an antigen for the production of a sequence-specific antiserum. In a radioimmunoassay, peptides that extend toward the amino terminal from Ala34, such as [Tyr,Gly]-ACTH34-39, ACTH18-39, and ACTH, had greater affinity for the antibody, which suggests that the antiserum recognizes the peptide bond preceding the alanyl residue. The assay readily detects 30 to 50 pmol of ACTH per liter with an incubation of only 3 h, and the antiserum cross reacts with larger molecular mass forms of the hormone. The amount of immunoreactive ACTH extracted by adsorption onto silicic acid from rat and human plasma was only 0.36 to 0.79 of that detected by a mid-region ACTH assay, which suggests proteolytic degradation at the carboxyl terminus of ACTH.

Infection of BSC-1 cells by SV40 brings about an increase of 7--11-fold in DNA polymerase activity, found in the nuclei and cytoplasm, respectively. The overall ratio between activites of DNA polymerase beta (3.1S) and DNA polymerase alpha (5.5S) remains fairly constant throughout infection. However,there is a large increase in DNA polymerase alpha2 (7.1S) in the cytoplasm, and its appearance in the nuclei late in infection. The addition of 1 M NaCl to infected cytoplasm,causes an aggregation of DNA polymerase alpha into a higher sedimenting form (9.8S), termed DNA polymerase alpha3. DNA polymerase alpha1, alpha2 and alpha3 are different molecular forms of the same enzyme, as can be seen by their similar inhibition by N-ethyl-maleimide, heparin and NaCl. However, this new activity, alpha3, is stimulated by dithiothreitol to a greater extent at pH 9.30 than at pH 7.94. The conformational changes induced in DNA polymerase and its increase in activity during infection with SV40 are discussed.

Systematic substitution of the natural L-amino acids in neurotensin by their D isomers reveals that the COOH-terminal portion of this tridecapeptide is required for binding to mast cell receptors: D-amino acid replacements from Pro10 through Leu13 substantially decrease that binding. Either blockage of the COOH-terminal carboxyl group as with N-methylamidation, or formation of a cyclic structure by the inclusion of a disulfide bond, a Cys2,13 substitution, markedly reduces the specific binding to mast cell receptor sites. Modifications in the NH2-terminal portion of neurotensin do not affect the binding to mast cells. However, D-Arg8 and D-Arg9 substitutions increase binding by factors of 5- to 6-fold. The hydroxyl group at position 3 or 11 is not essential for binding since Phe3 or Phe11 is equivalent to Tyr3 or Tyr11. The COOH-terminal penta- and hexapeptides are able to displace approximately 70% 125I-neurotensin relative to the intact peptide. Of 18 other biologically active peptides tested, only xenopsin, a naturally occurring COOH-terminal analog of neurotensin, and bradykinin effectively compete in the binding assay to an extent of 60 and 100%, respectively. Histamine, diphenhydramine, and noradrenaline are ineffective in this regard.

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.

Parallel in vitro bioassays using rat uterus and guinea pig large intestine tissues specific for the bombesin family of peptides, demonstrated that the bombesin-like peptides present in bovine milk can produce a dose-related response similar to bombesin and litorin. The bioactivity of this type of milk peptide appeared to be approximately 20-50% as active as the amphibian peptides. These data support the proposal that a bombesin immunoreactive peptide in milk contains bombesin bioactivity.