A practical guide to data management and sharing for biomedical laboratory researchers.

Effective data management and sharing have become increasingly crucial in biomedical research; however, many laboratory researchers lack the necessary tools and knowledge to address this challenge. This article provides an introductory guide into research data management (RDM), and the importance of FAIR (Findable, Accessible, Interoperable, and Reusable) data-sharing principles for laboratory researchers produced by practicing scientists. We explore the advantages of implementing organized data management strategies and introduce key concepts such as data standards, data documentation, and the distinction between machine and human-readable data formats. Furthermore, we offer practical guidance for creating a data management plan and establishing efficient data workflows within the laboratory setting, suitable for labs of all sizes. This includes an examination of requirements analysis, the development of a data dictionary for routine data elements, the implementation of unique subject identifiers, and the formulation of standard operating procedures (SOPs) for seamless data flow. To aid researchers in implementing these practices, we present a simple organizational system as an illustrative example, which can be tailored to suit individual needs and research requirements. By presenting a user-friendly approach, this guide serves as an introduction to the field of RDM and offers practical tips to help researchers effortlessly meet the common data management and sharing mandates rapidly becoming prevalent in biomedical research.

Locomotion after spinal cord injury depends on constitutive activity in serotonin receptors.

Following spinal cord injury (SCI) neurons caudal to the injury are capable of rhythmic locomotor-related activity that can form the basis for substantial functional recovery of stepping despite the loss of crucial brain stem-derived neuromodulators like serotonin (5-HT). Here we investigated the contribution of constitutive 5-HT(2) receptor activity (activity in the absence of 5-HT) to locomotion after SCI. We used a staggered hemisection injury model in rats to study this because these rats showed a robust recovery of locomotor function and yet a loss of most descending axons. Immunolabeling for 5-HT showed little remaining 5-HT below the injury, and locomotor ability was not correlated with the amount of residual 5-HT. Furthermore, blocking 5-HT(2) receptors with an intrathecal (IT) application of the neutral antagonist SB242084 did not affect locomotion (locomotor score and kinematics were unaffected), further indicating that residual 5-HT below the injury did not contribute to generation of locomotion. As a positive control, we found that the same application of SB242084 completely antagonized the muscle activity induced by exogenous application of the 5-HT(2) receptor agonists alpha-methyl-5-HT (IT). In contrast, blocking constitutive 5-HT(2) receptor activity with the potent inverse agonist SB206553 (IT) severely impaired stepping as assessed with kinematic recordings, eliminating most hindlimb weight support and overall reducing the locomotor score in both hind legs. However, even in the most severely impaired animals, rhythmic sweeping movements of the hindlimb feet were still visible during forelimb locomotion, suggesting that SB206553 did not completely eliminate locomotor drive to the motoneurons or motoneuron excitability. The same application of SB206553 had no affect on stepping in normal rats. Thus while normal rats can compensate for loss of 5-HT(2) receptor activity, after severe spinal cord injury rats require constitutive activity in these 5-HT(2) receptors to produce locomotion.

Transplantation and repair: combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury.

STUDY DESIGN: Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats. OBJECTIVES: To report effects on bladder function following the administration of a combined treatment for complete SCI. SETTING: University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, Canada. METHODS: Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks. RESULTS: Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III). CONCLUSION: We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rat's ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.

Evidence for renal kinins as mediators of amino acid-induced hyperperfusion and hyperfiltration in the rat.

This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.

BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats.

Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.

A fluorometric study of the interaction of bradykinin with lipids.

The interaction of bradykinin (BK) with lipids has been followed by steady-state fluorescence measurements. Addition of either cerebroside sulfate (CS) or phosphatidylinositol (PI), solubilized with the nonionic surfactant C12E8, to BK or its analogue [Gly6]-BK enhances the relative fluorescence intensity of peptide emission at 288 nm. Fluorometric titration of the peptide with lipid has been used to quantitate the interactions in terms of stoichiometry and equilibrium constant. Job's method of continuous variation for the BK-CS interaction gave a stoichiometry of 1:2 for the complex. The value of the equilibrium constant, K, for the interaction of either BK or [Gly6]-BK with CS is 1.5.10(4) M-1. The BK-PI interaction is weaker; K = 5.0.10(3) M-1. Although electrostatic forces no doubt play a major role in these interactions, measurements on the model peptide Gly-Phe-Gly indicate that the phenylalanine residues of BK are disposed in the hydrophobic environment provided by the lipid-C12E8 mixed micelle. 13C-NMR measurements on [99% 13C alpha-Gly6]-BK show that there is no change in its cis/trans ratio upon interaction with CS. The increase in the relative fluorescence intensity of BK accompanying its cooperative interaction with sodium dodecyl sulfate (SDS) implicates the role of hydrophobic forces in this interaction as well. These results bear on the interpretation of the changes in circular dichroism (CD) of BK caused by SDS.

Bradykinin-induced burst of prostaglandin formation in osteoblasts is mediated via B2 bradykinin receptors.

The receptor subtype mediating the bradykinin (Bk)-induced burst of prostaglandin formation in osteoblasts has been studied. Bk, but not des-Arg9-Bk, induced the formation of prostaglandin E2 and prostacyclin in neonatal mouse calvarial bones incubated for 30 minutes. Bk-induced prostaglandin synthesis is neonatal mouse calvarial bones was significantly inhibited by the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk. The B2 Bk receptor agonists Bk and Lys-Bk, but not the B1 Bk receptor agonist des-Arg9-Bk, caused a rapid burst (5 minutes) of prostaglandin E2 and prostacyclin formation in isolated osteoblast-like cells from neonatal mouse calvarial bones and in the murine osteoblastic cell lineage MC3T3-E1. When comparing the relative potency of different kinin analogs on their stimulatory effect on prostaglandin formation in isolated osteoblast-like cells and in MC3T3-E1 cells, we found that Bk = Lys-Bk greater than Met-lys-Bk much much greater than Des-Arg9-Bk. Bk-induced prostaglandin synthesis in isolated osteoblast-like cells and in MC3T3-E1 cells was inhibited by D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk, whereas the B1 Bk receptor antagonist des-Arg9-Leu8-Bk had no effect. Total binding of 3H-Bk (3-20 nM; 4 degrees C) to whole MC3T3-E1 cells reached a maximum after 4-5 h. An excess of nonradioactive Bk (1 microM) reduced cell-associated radioactivity by 20-30%. The B2 Bk receptor agonist Bk and the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk were able to reduce specific binding, but the B1 Bk receptor agonist des-Arg9-Bk was unable to reduce the specific binding of 3H-Bk to whole MC3T3-E1 cells. These findings indicate the presence of B2 Bk receptors on osteoblasts coupled to enhanced prostaglandin synthesis.

Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts.

IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.

Vasodepressor role of endogenous bradykinin assessed by a bradykinin antagonist.

This study was designed to examine the contribution of bradykinin to the depressor effect of different antihypertensive drugs in two-kidney renovascular hypertensive rats, using a new specific antagonist of bradykinin. First, the inhibitory capacity of this peptide for exogenously injected bradykinin (75-200 ng) was tested. An inhibition of the vasodepressor action of bradykinin by over 50% was found when the bradykinin inhibitor was infused at a rate of 40 micrograms/min, with little difference at higher rates of infusion. This inhibitor then was infused in three groups of renovascular hypertensive rats after their blood pressure had been decreased by pretreatment with the converting enzyme inhibitor enalapril (MK 421), saralasin, or sodium nitroprusside, respectively. Infusion of the inhibitor produced an immediate 30% increase in blood pressure only in the enalapril-treated group. These results indicate that bradykinin is involved in the decrease of blood pressure produced by converting enzyme inhibition in experimental renovascular hypertension.

New synthetic antagonists of bradykinin.

1 A new synthetic bradykinin analogue was found to be an antagonist of bradykinin-induced vascular permeability in rabbit skin. It was effective in equimolar concentrations. 2 These analogues also antagonized the action of bradykinin in contracting the guinea-pig isolated ileum. The mean pA2 values of five different antagonists ranged from 5.3-6.4 respectively, on this preparation. 3 Our observations, together with those of others suggest that these antagonists act on the same receptor types, viz., B2, in rabbit blood vessels and in smooth muscle of guinea-pig ileum. 4 Our results support the view that the way is now promising for the synthesis of potent specific antagonists of bradykinin for experimental and therapeutic use.

Characterization of receptor-mediated actions of T-kinin.

T-Kinin (Ile-Ser-bradykinin) is unique to the rat. This study characterizes the receptors involved in T-kinin activity on both the intact isolated rat uterus and membrane receptor preparations of the rat uterus. The results show that T-kinin acts through kinin B2 receptors in the rat uterus as demonstrated by B2 receptor-antagonist inhibition. While the potency of T-kinin on rat uterus contraction was similar to that of bradykinin, binding studies showed that the affinity of T-kinin to the receptor was 10-fold lower than that of bradykinin. On the other hand, the D isomer of T-kinin, D-Ile-Ser-bradykinin, had an affinity for the receptor greater than that of T-kinin and was more potent in causing contraction. Comparing this finding with our previously published report that D-Ile-Ser-bradykinin is not active on the kinin receptor for vascular permeability indicates that the kinin receptors in the rat uterus are not the same as those previously reported in the smooth muscle of the vasculature, i.e. there exists subclasses of kinin B2 receptors. The data from binding studies on a variety of T-kinin analogues show that the substitution of hydroxyproline (Hyp) for Pro5, together with the D-configuration at Ile1 and/or Ser2 may be useful for the development of selective T-kinin antagonists. Studies involving pretreatment of the tissue with indomethacin demonstrated that prostaglandin release was more of a component of T-kinin's activity on the rat uterus than that of bradykinin.

Interactions of kinins with angiotensin I converting enzyme (kininase II).

Angiotensin I converting enzyme (ACE) was purified to homogeneity from porcine kidney in order to determine whether iodobradykinins bind to the enzyme and, if so, whether pGlu-Trp-Pro-Arg-Pro-Gin-Ile-Pro-Pro, SQ20881, a competitive ACE inhibitor, changes the conformation of the enzyme in such a way that it binds kinins with an affinity and specificity expected of a bradykinin (BK) receptor, i.e. where the BK potentiating action of SQ20881 involves an increase in the number of BK receptors due to a conformational change in ACE. 125I-Labeled derivatives of [Tyr1]-kallidin and [Tyr-8]-bradykinin bound to the EDTA-inhibited enzyme, and binding was inhibited by nonradioactive BK. [125I-Tyr5]-BK was not bound by the enzyme. Specificity of [125I-Tyr5]-kallidin (T1K) binding was tested with forty-eight BK analogs, and the concentrations of analogs that inhibited 50% of T1K binding were determined. BK at 1.6 +/- 0.3 X 10(-8) M inhibited 505 of T1K binding. In addition, the concentrations of analogs that decreased by 50% the rate of [3H]-Hip-Gly-Gly ([3H]-HGG) hydrolysis by ACE were assessed. BK at 1.2 +/- 0.2 X 10(-6) M decreased the rate of [3H]-HGG hydrolysis by 50%. A comparison between these concentrations of analogs for inhibition of T1K binding and [3H]-HGG hydrolysis yielded a high correlation coefficient (r = 0.85). The specificity of ACE binding was clearly different from that expected of a BK receptor. Compounds structurally unrelated to BK, such as 5Q20881, pGlu-Lys-Trp-Ala-Pro-OH (BPP5a) and angiotensin I, inhibited T1K binding and [3H]-HGG hydrolysis by ACE.

Further studies of myometrial bradykinin receptor-like binding.

[125I-Tyr1]Kallidin (T1K), a bradykinin (BK) analog with biological potency comparable to BK, was used as a probe for BK receptor-like binding from bovine uterine myometrium. BK binding exhibited a high affinity, Kdissoc = 1.65 X 10(-10) M. The specificity of T1K binding was examined with forty-four BK analogs. Comparison of the binding inhibitory potencies with the relative biological potencies of these analogs on isolated rat uterus resulted in a good correlation, r = 0.87. BK binding activity was solubilized with CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent. The solubilized binding activity exhibited a BK binding affinity, Kdissoc = 2.25 X 10(-10) M, and a specificity for three 125I-labeled kinins similar to those of the particulate BK receptor-like binding activity.

Induction of histamine release from human skin mast cells by bradykinin analogs.

Kinins are potent proinflammatory peptides that induce histamine release from rodent mast cells. We examined the ability of bradykinin, lysylbradykinin and a series of kinin analogs to cause histamine release from human basophils, human lung mast cells and human skin mast cells. At concentrations ranging from 0.1 microM to 1 mM, bradykinin failed to cause histamine release from any of the human histamine-containing cells studied. Lysylbradykinin was also without effect on basophils and lung mast cells, but was a weak secretagogue for human skin mast cells, inducing 5.5 +/- 3% (mean +/- SD) of total cellular histamine release at a concentration of 10(-5) M. Similarly, when sixteen recently developed bradykinin antagonists were examined, these compounds had no effect on basophils or lung mast cells but all sixteen induced dose-dependent histamine release from skin mast cells. The release process was temperature dependent and, at a concentration of 10(-5) M, the antagonists induced 8-27% histamine release. Although preincubation of cells with 10(-3) M bradykinin or des(Arg9) bradykinin significantly inhibited antagonist-induced histamine release, the requirement for such high concentrations of these peptides to cause inhibition suggested that histamine release is not mediated by either B1 or B2 kinin receptors. To understand further the mechanism of histamine release, we examined a series of bradykinin analogs with single amino acid substitutions in the bradykinin sequence. Replacement of proline7 in the bradykinin sequence with D-phenylalanine is the essential change used to convert kinin analogs into antagonists, and 10(-5) M [DPhe7]-bradykinin induced 8-10% histamine release. Other analogs, devoid of antagonist activity, however, such as [DPhe6]-bradykinin and [LPhe7]-bradykinin were able to induce equivalent levels of histamine release. The ability to induce histamine release appears to be related, at least in part, to aromaticity, since [DTrp6]-bradykinin and [DTrp7]-bradykinin induced greater amounts of histamine release than equivalent [DPhe]-analogs, causing approximately 20% histamine release at 10(-5) M. By contrast, [DAla7]-bradykinin was an ineffective stimulus. In summary, a single amino acid substitution can convert bradykinin into a secretagogue for human skin mast cells. The ability of kinin analogs to induce histamine release from skin mast cells, but not lung mast cells or basophils, emphasizes the heterogeneity of human histamine-containing cells.

Bradykinin analogs containing alpha-aminoisobutyric acid (Aib).

All seven possible bradykinin (BK) analogs contained Aib in place of proline have been synthesized by the solid phase method and assayed for in vitro myotropic activity on the guinea pig ileum and rat uterus, and in vivo on the rat blood pressure, both in intravenous and intra-aortic administration. [Aib(2,3)]-BK, [Aib(2,7)]-BK, and [Aib(2,3,7)]-BK had no in vivo or in vitro activities; [Aib(2)]-BK, [Aib(3)]-BK and [Aib(3,7)]-BK had moderate BK-like activities and a significantly increased resistance to pulmonary inactivation in the rat ([Aib(3,7)]-BK was totally resistant). [Aib(7)]-BK was found to be the most active position seven BK analog yet assayed on the rat blood pressure, and shows remarkably high ileum (4 times BK) and intravenous rat blood pressure (6 times BK) activity.

Competitive antagonists of bradykinin.

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.

The inhibition of glandular kallikrein by peptide analog antagonists of bradykinin.

Bradykinin sequence analog receptor antagonists exhibit at their carboxyl termini features which contribute to the affinity of peptide inhibitors of glandular kallikreins. These features include a preference for L-Arg over L-Lys at position P1 and bulky D-amino acids at P3. There is minimal steric restriction at P2. Three representative receptor antagonists were examined for their capacity to inhibit the amidolytic activity of human urinary kallikrein (HUK). The Ki values for B4307 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg), B4308 (Lys-Lys-Arg-Hyp-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg), and B3852 (Arg-Pro-Hyp-Gly-Phe-Ser-DPhe-Phe-Arg) are 0.5, 0.3 and 2.5 microM, respectively. B4308 and B3852 were also shown to inhibit rat urinary kallikrein with Ki's of 1.0 and 5.5 microM. In the estrous rat uterus assay, 0.4 to 1.6 microM quantities of B4308 gave 5 to 10 times as much inhibition of contractile activity when added at the beginning of the incubation of HUK with human low molecular weight kininogen than when added upon addition of the mixture to the organ bath. These antagonists may inhibit the kallikrein-kinin system not only by blocking the binding of kinins to their receptor(s) but also by inhibiting the release of kinins from kininogens.

Interaction of bradykinin with sodium dodecyl sulfate and certain acidic lipids.

The striking change in the circular dichroism (CD) of bradykinin (BK) occasioned by its interaction with sodium dodecyl sulfate (SDS) is evidently due in large part to a change in the conformation of the C-terminal tetrapeptide moiety of the hormone. The full change in CD is induced by the binding of two molecules of monomeric SDS per peptide molecule, the complex being aggregated. Formation of the 1:2 BK-SDS complex apparently proceeds via intermediates of stoichiometry 1:1 and 2:1. The cooperative nature of the interaction is attributed to the SDS-promoted aggregation of BK. Electrostatic interactions with the Arg residues appear important for the binding reaction per se. CD reveals that BK also interacts with acidic lipids which bear a net electrical charge (e.g., cerebroside sulfate and phosphatidyl inositol) but not with lipids bearing no net charge (e.g., cerebroside and phosphatidyl choline). The interactions are with particular mixed micelles of the lipid and the nonionic surfactant used for their solubilization, micellar size and structure being examined by surface tensiometry and electron microscopy.

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.

Raman studies on bradykinin and a cyclic bradykinin.

Laser Raman spectra of bradykinin in water, deuterium oxide, and the solid phase were recorded. From the spectra it was concluded that bradykinin conformation is comprised of ordered and unordered structure. The ordered structure appears to be some form of reverse turn. Furthermore, it seems that there is an enhancement of the turn structure in the solid phase. A cyclic cystine containing analog of bradykinin was also examined with Raman spectroscopy. The cyclic bradykinin analog gives a Raman spectrum very similar to that of the linear bradykinin and therefore must share similar conformational forms with bradykinin. The restrictive Cys-Cys disulfide in the cyclic bradykinin must serve to maintain a conformation acceptable to bradykinin receptors since the cyclic peptide exhibits biological activity.