The Absence of the ACE N-Domain Decreases Renal Inflammation and Facilitates Sodium Excretion during Diabetic Kidney Disease.

BACKGROUND: Recent evidence emphasizes the critical role of inflammation in the development of diabetic nephropathy. Angiotensin-converting enzyme (ACE) plays an active role in regulating the renal inflammatory response associated with diabetes. Studies have also shown that ACE has roles in inflammation and the immune response that are independent of angiotensin II. ACE's two catalytically independent domains, the N- and C-domains, can process a variety of substrates other than angiotensin I. METHODS: To examine the relative contributions of each ACE domain to the sodium retentive state, renal inflammation, and renal injury associated with diabetic kidney disease, we used streptozotocin to induce diabetes in wild-type mice and in genetic mouse models lacking either a functional ACE N-domain (NKO mice) or C-domain (CKO mice). RESULTS: In response to a saline challenge, diabetic NKO mice excreted 32% more urinary sodium compared with diabetic wild-type or CKO mice. Diabetic NKO mice also exhibited 55% less renal epithelial sodium channel cleavage (a marker of channel activity), 55% less renal IL-1ß, 53% less renal TNF-α, and 53% less albuminuria than diabetic wild-type mice. This protective phenotype was not associated with changes in renal angiotensin II levels. Further, we present evidence that the anti-inflammatory tetrapeptide N-acetyl-seryl-asparyl-lysyl-proline (AcSDKP), an ACE N-domain-specific substrate that accumulates in the urine of NKO mice, mediates the beneficial effects observed in the NKO. CONCLUSIONS: These data indicate that increasing AcSDKP by blocking the ACE N-domain facilitates sodium excretion and ameliorates diabetic kidney disease independent of intrarenal angiotensin II regulation.

Effects of MicroRNA-494 on Astrocyte Proliferation and Synaptic Remodeling in the Spinal Cord of a Rat Model of Chronic Compressive Spinal Cord Injury by Regulating the Nogo/Ngr Signaling Pathway.

BACKGROUND/AIMS: Chronic compression of the spinal cord causes the loss of motor neurons in the anterior horn, but the precise and extensive mechanism for the loss is not completely determined. Therefore, this study aims to explore the role of microRNA-494 (miR-494) in the proliferation of astrocytes and in the synaptic remodeling in the spinal cord of a rat model of chronic spinal cord injury (SCI) by regulating the Nogo/NgR signaling pathway. METHODS: A rat model of chronic, compressive SCI was established, and the spinal cord state, blood supply changes, and astrocyte apoptosis were observed. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect expression of miR-494 and the Nogo/NgR signaling pathway-related genes. Fluorescence in situ hybridization (FISH) was used for detecting miR-494 expression and distribution. RESULTS: Higher miR-494 expression was accompanied by the inhibition of astrocyte proliferation and synaptic remodeling. In addition, CDK6 could be regulated by miR-494 and was shown to be one of the target genes of miR-494. Positive expression of miR-494 detected by FISH was consistent with the results from RT-qPCR that miR-494 could downregulate CDK6 gene expression. Moreover, the direct miR-494 target CDK6 plays important inhibitory roles in chronic SCI by suppressing the Nogo/ NgR signaling pathway. CONCLUSIONS: The results demonstrated that miR-494 inhibition can promote astrocyte proliferation and synaptic remodeling by suppressing the Nogo/NgR signaling pathway in a rat model of chronic SCI.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo.

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Ligand Strain and Entropic Effects on the Binding of Macrocyclic and Linear Inhibitors: Molecular Modeling of Penicillopepsin Complexes.

Using extensive molecular dynamics simulations, we investigate the structure and dynamics of the complexes formed between penicillopepsin and two peptidomimetic inhibitors: a linear compound, isovaleryl(P4)-valine(P3)-asparagine(P2)-leucine(P1)-phosphonate-phenylalanine(P1'), and its macrocylic analog that includes a methylene bridge between the Asn(P2) and Phe(P1') side chains. The macrocyclic inhibitor, which has a 420-fold stronger affinity than that of the acyclic one, has been considered to lower the entropic penalty for binding. To better understand this binding preference, the solution structure of the inhibitors is studied by molecular dynamics simulations. Subsequently, we assess the influence of the enzyme/inhibitor contacts, the enzyme-induced inhibitor strain, the variation of the ligand configurational entropy and the enzyme reorganization by combining molecular-mechanics Poisson-Boltzmann surface area and normal mode calculations with conformational entropy calculations. We find that there is no relevant entropic stabilization on the binding of the cyclic inhibitor with respect to the acyclic analog because the methylene bridge does not reduce appreciably the conformational flexibility of the free inhibitor. The most important factors explaining the stronger affinity of the macrocyclic inhibitor are the conformational filtering and the lower ligand strain induced by the methylene bridge.

Total synthesis and biological investigation of (-)-promysalin.

Compounds that specifically target pathogenic bacteria are greatly needed, and identifying the method by which they act would provide new avenues of treatment. Herein we report the concise, high-yielding total synthesis (eight steps, 35% yield) of promysalin, a natural product that displays antivirulence phenotypes against pathogenic bacteria. Guided by bioinformatics, four diastereomers were synthesized, and the relative and absolute stereochemistries were confirmed by spectral and biological analysis. Finally, we show for the first time that promysalin displays two antivirulence phenotypes: the dispersion of mature biofilms and the inhibition of pyoverdine production, hinting at a unique pathogenic-specific mechanism of action.

Tuning cobalt(III) Schiff base complexes as activated protein inhibitors.

Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation. Fluorescent imidazoles (C3Im) were prepared as axial ligands in [Co(acacen)(L)2](+) to produce complexes (CoC3Im) that could report on ligand exchange and, thus, complex stability. These fluorescent imidazole reporters guided the design of a new dinuclear Co(III) Schiff base complex containing bridging diimidazole ligands, which exhibits enhanced stability to ligand exchange with competing imidazoles and to hydrolysis within a biologically relevant pH range. These studies inform the design of biocompatible Co(III) Schiff base complexes that can be selectively activated for protein inhibition with spatial and temporal specificity.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis.

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.

RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis.

The integrin α(v)ß(3) plays an important role in angiogenesis. It is expressed on tumoral endothelial cells as well as on some tumor cells. RGD peptides are well-known to bind preferentially to the α(v)ß(3) integrin. In this context, targeting tumor cells or tumor vasculature by RGD-based strategies is a promising approach for delivering anticancer drugs or contrast agents for cancer therapy and diagnosis. RGD-based strategies include antagonist drugs (peptidic or peptidomimetic) of the RGD sequence, RGD-conjugates, and the grafting of the RGD peptide or peptidomimetic, as targeting ligand, at the surface of nanocarriers. Although all strategies are overviewed, this review aims to particularly highlight the position of RGD-based nanoparticles in cancer therapy and imaging. This review is divided into three parts: the first one describes the context of angiogenesis, the role of the integrin α(v)ß(3), and the binding of the RGD peptide to this integrin; the second one focuses on RGD-based strategies in cancer therapy; while the third one focuses on RGD-based strategies in cancer diagnosis.

Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A.

Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

Transport of hepcidin, an iron-regulatory peptide hormone, into retinal pigment epithelial cells via oligopeptide transporters and its relevance to iron homeostasis.

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC50 values in the range of 0.4-1.7 µM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor antagonists based on small bifunctional templates: arginine and phenylalanine side chain groups are keys for receptor activity.

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1). These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 microM) inhibited both TFLLR-amide (10 microM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 microM) calcium signaling in a cultured human HEK cell assay.

Polymeric tubulysin-peptide nanoparticles with potent antitumor activity.

PURPOSE: Tubulysins are naturally occurring tetrapeptides with potent antiproliferative activity against multiple cancer cell lines. However, they are also highly toxic in animal models. In order to improve the therapeutic index of this class of compounds, a nanoparticle prodrug of tubulysin A (TubA) was synthesized and evaluated in vitro and in vivo. EXPERIMENTAL DESIGN: A thiol derivative of TubA was covalently attached to a linear, beta-cyclodextrin based polymer through a disulfide linker (CDP-TubA). The polymer conjugate assembled into stable nanoparticles. Inhibition of tubulin polymerization and antiproliferative activity of the polymer conjugate were evaluated in vitro. The preclinical efficacy of CDP-TubA administered i.v. was evaluated in nude mice bearing s.c. implanted human HT29 colorectal and H460 non-small cell lung carcinoma tumors. RESULTS: The IC(50) of CDP-TubA (in Tub A equivalents) was 24, 5, and 10 nmol/L versus 3, 1, and 2 nmol/L for Tub A in NCI-H1299 (lung), HT-29 (colon), and A2780 (ovarian) cell lines, respectively. Tub A and the active thiol derivative were potent inhibitors of tubulin polymerization, whereas CDP-TubA showed minimal inhibition, indicating that target inhibition requires release of the peptide drug from the nanoparticles. The maximum tolerated dose of CDP-TubA was 6 mg/kg (in TubA equivalents) versus 0.05 mg/kg for TubA in nude mice. In vivo, a single treatment cycle of three weekly doses of CDP-TubA showed a potent antitumor effect and significantly prolonged survival compared with TubA alone. CONCLUSIONS: Cyclodextrin polymerized nanoparticles are an enabling technology for the safe and effective delivery of tubulysins for the treatment of cancer.

A new perspective in sepsis treatment: could RGD-dependent integrins be novel targets?

Sepsis is a life-threatening condition caused by the response of the body to an infection, and has recently been regarded as a global health priority because of the lack of effective treatments available. Vascular endothelial cells have a crucial role in sepsis and are believed to be a major target of pathogens during the early stages of infection. Accumulating evidence suggests that common sepsis pathogens, including bacteria, fungi, and viruses, all contain a critical integrin recognition motif, Arg-Gly-Asp (RGD), in their major cell wall-exposed proteins that might act as ligands to crosslink to vascular endothelial cells, triggering systemic dysregulation resulting in sepsis. In this review, we discuss the potential of anti-integrin therapy in the treatment of sepsis and septic shock.

Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function.

Pseudomonas aeruginosa: causes serious infections in patients with compromised immune systems and exhibits resistance to multiple antibiotics. The rising threat of antimicrobial resistance means that new methods are necessary for treating microbial infections. We conducted a high-throughput screen for compounds that can quench the innate fluorescence of the chromophore region of the P. aeruginosa siderophore pyoverdine, a key virulence factor. Several hits were identified that effectively quench pyoverdine fluorescence, and two compounds considerably improved the survival of Caenorhabditis elegans when worms were challenged with P. aeruginosa. Commercially available analogs of the best hit, PQ3, were tested for their ability to rescue C. elegans from P. aeruginosa and to interact with pyoverdine via fluorescence and solution NMR spectroscopy. 1H-15N and 1H-13C HSQC NMR were used to identify the binding site of PQ3c. The structure model of pyoverdine in complex with PQ3c was obtained using molecular docking and molecular dynamics simulations. PQ3c occupied a shallow groove on pyoverdine formed by the chromophore and N-terminal residues of the peptide chain. Electrostatic interactions and π-orbital stacking drove stabilization of this binding. PQ3c may serve as a scaffold for the development of pyoverdine inhibitors with higher potency and specificity. The discovery of a small-molecule binding site on apo-pyoverdine with functional significance provides a new direction in the search of therapeutically effective reagent to treat P. aeruginosa infections. Abbreviations: NMR: nuclear magnetic resonance; SAR: structure-activity relationship; MD: molecular dynamics; RMSF: root-mean-square fluctuation; HSQC: heteronuclear single quantum correlation; DMSO: dimethyl sulfoxide; Δδavg: average amide chemical shift change; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; RMSD: root-mean-square deviation; LJ-SR: Lennard-Jones short-range; Coul-SR: Coulombic short-range; FRET: fluorescence resonance energy transfer.

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury.

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-ß at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

An analog of enkephalin having prolonged opiate-like effects in vivo.

Intraventricular administration of the enkephalin analog, [D-Ala2]-metenkephalin, induces profound and long-lasting analgesia, as well as other opiate-like behavioral effects in the rat. This analgesia was highly dose dependent, of much greater magnitude, and about 30 times longer lasting than that produced by the naturally occurring peptide, methionine-enkephalin. The behavioral effects of the [D-Ala2] analog could be completely reversed by the opiate antagonist, naloxone, suggesting that these effects were mediated by opiate receptors. Systemic administration of naloxone alone resulted in a significant increase in pain sensitivity. These findings support the view that endogenous opiate systems may play an important role in modulating pain sensitivity.

Derivatives of 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid as novel fibrinogen receptor antagonists.

It has been proposed a novel method for obtaining of 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid as Arg-mimetic within the framework of search for novel fibrinogen receptor antagonists. New alpha (IIb)beta(3) antagonists were prepared on a base of 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid. Their high antiaggregatory activities in a human platelet rich plasma and ability to block FITC-Fg binding to alpha (IIb)beta(3) on washed human platelets were estimated.