Free Acid Analog Research Articles

Thin-skinned intrinsically defect-free asymmetric mono-esterified hollow fiber precursors for crosslinkable polyimide gas separation membranes

Engineering of spinning solution compositions, conditions and polymer molecular weight is described to create intrinsically defect-free thin-skinned precursors for high performance ester-crosslinked asymmetric hollow fiber membranes. The ultimate goal is to crosslink the precursors to provide robust performance for an aggressive natural gas feed. To provide insights free from CO2-induced changes in properties in the uncrosslinked samples, studies with less interacting O2, N2 and He feeds were also used to assess properties. The combined analyses indicate an absence of substructure resistance within the hollow fibers and an effective selective layer between 0.1 and 0.14µm. This thickness represents a valuable reduction over previously achievable values for this so-called propane-diol monoesterified crosslinkable polyimide (PDMC) polymer in defect-free asymmetric form, which were previously in the range of 0.5µm. Indeed, the significantly reduced skin layer thickness offers the potential to improve the gas separation productivity by 3.6X if this advantage can be carried forward into the ultimate crosslinked fibers. Additional comparisons to the free acid form of the precursor (without esterification) show advantages to the current less hydrophilic precursor in terms of ability to create a thinner precursor selective layer prior to crosslinking than for the free acid form. To date, the free acid analog to PDMC has only been able to be created with a 0.7µm selective layer vs. the current 0.1–0.14µm skin reported here. Optimization of the crosslinking process for both precursors is beyond the scope of this work, since it is itself, complex and still underway.

18F-FDG Cardiac Studies for Identifying Ischemic Memory

Myocardial ischemia is associated with profound changes in substrate utilization by the myocardium for energy production. This change occurs with the onset of ischemia, but persists much longer, perhaps for several hours or even days after an episode of myocardial ischemia, which is termed ischemic memory. Recently, radiolabeled glucose analogue (Fluorine-18 labeled deoxyglucose, 18FDG) and free acid analogue (β-methyl-p-123I-iodophenyl-pentadecanoic acid, 123I-BMIPP) have been investigated to detect myocardial ischemia in stable coronary artery disease (CAD) or suspected acute coronary syndrome (ACS). These studies demonstrate that BMIPP imaging holds promise for evaluation of patients with suspected ACS in the emergency department. Exercise 18FDG imaging offers several potential advantages over conventional stress-rest perfusion imaging for the detection of CAD. Furthermore, ischemia-induced 18FDG uptake may persist for several to 24 hours in patients with stress-induced myocardial ischemia. Therefore, 18FDG may also be a useful memory marker in detection of antecedent myocardial ischemia in suspected ACS. However, some important issues about 18FDG ischemic imaging need to be further investigated. In this article the advances of myocardial ischemic imaging with 18FDG are reviewed. Several unresolved issues and the perspective of this modality are also discussed.

Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects.

Mast cells are involved in atopic disorders, often exacerbated by stress, and are located perivascularly close to sympathetic and sensory nerve endings. Mast cells are activated by electrical nerve stimulation and millimolar concentrations of neuropeptides, such as substance P (SP). Moreover, acute psychological stress induces CRH-dependent mast cell degranulation. Intradermal administration of rat/human CRH (0.1-10 microM) in the rat induced mast cell degranulation and increased capillary permeability in a dose-dependent fashion. The effect of CRH on Evans blue extravasation was stronger than equimolar concentrations of the mast cell secretagogue compound 48/80 or SP. The free acid analog of CRH, which does not interact with its receptors (CRHR), had no biological activity. Moreover, systemic administration of antalarmin, a nonpeptide CRHR1 antagonist, prevented vascular permeability only by CRH and not by compound 48/80 or SP. CRHR1 was also identified in cultured leukemic human mast cells using RT-PCR. The stimulatory effect of CRH, like that of compound 48/80 on skin vasodilation, could not be elicited in the mast cell deficient W/Wv mice but was present in their +/+ controls, as well as in C57BL/6J mice; histamine could still induce vasodilation in the W/Wv mice. Treatment of rats neonatally with capsaicin had no effect on either Evans blue extravasation or mast cell degranulation, indicating that the effect of exogenous CRH in the skin was not secondary to or dependent on the release of neuropeptides from sensory nerve endings. The effect of CRH on Evans blue extravasation and mast cell degranulation was inhibited by the mast cell stabilizer disodium cromoglycate (cromolyn), but not by the antisecretory molecule somatostatin. To investigate which vasodilatory molecules might be involved in the increase in vascular permeability, the CRH injection site was pretreated with the H1-receptor antagonist diphenhydramine, which largely inhibited the CRH effect, suggesting that histamine was involved in the CRH-induced vasodilation. The possibility that nitric oxide might also be involved was tested using pretreatment with a nitric oxide synthase inhibitor that, however, increased the effect of CRH. These findings indicate that CRH activates skin mast cells at least via a CRHR1-dependent mechanism leading to vasodilation and increased vascular permeability. The present results have implications for the pathophysiology and possible therapy of skin disorders, such as atopic dermatitis, eczema, psoriasis, and urticaria, which are exacerbated or precipitated by stress.

Conformational Preferences of the CalliFMRFamides and their Free-Acid Analogues

A molecular dynamics study was undertaken to determine the conformational basis for the differing activities of the insect neuropeptide hormones calliFMRFamide 3 (SPSQDFMRF-NH2). calliFMRFamide 5 (APGQDFMRF-NH2) and their corresponding free-acid analogues (SPSQDFM-RF-OH and APGQDFMRF-OH) in two insect bioassays. A simulated annealing protocol was used to determine the range of conformers available to the linear peptides. Analysis of the conformers obtained indicated that all the peptides exhibited distinct secondary structure preferences. These, when correlated with their biological activities, enabled the formulation of putative conformation-activity relationships for the peptides.

Structure-activity relationship study of R396, an NK 2 tachykinin antagonist selective for the NK 2B receptor subtype

We report on a structure-activity study of R396 (Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH 2), a linear hexapeptide tachykinin antagonist selective for the putative NK 2B receptor subtype. Asp 2, Trp 4 and the C-terminal glycinamide have been challenged by classical amino acid substitutions with the aim of elucidating the structural requirements responsible for NK 2 subtype selectivity. The biological activities indicate that Asp 2 has a crucial role for the high affinity of R396 at the NK 2B subtype: none of the analogues substituted in position 2 display higher affinity as compared to R396, regardless of the nature of the residue introduced. Trp 4 has been replaced by other aromatic residues, again yielding weak antagonist or inactive compounds. Finally, the C-terminal amide appears to be crucial for affinity, the free acid analogue being devoid of biological activity. On the other hand, antagonistic activity is maintained both by the desGly pentapeptide and by the analogue bearing βAla in place of Gly in position 6. In conclusion, since the NK 2B selectivity pattern was maintained throughout the whole series of R396 replacement analogues, we speculate that the overall conformational features of this family of linear hexapeptides favour the interaction with the NK 2B receptor subtype.

A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone.

We have identified an amino acid sequence within the E peptide of the insulin-like growth factor IB (IGF-IB) precursor that is biologically active and designated this peptide insulin-like growth factor IB-(103-124) E1 amide (IBE1). Its existence was predicted by a flanking Gly-Lys-Lys-Lys, a signal sequence for sequential proteolytic cleavage and peptidyl C-terminal amidation. A synthetic analog of the predicted IBE1 peptide, designated Y-23-R-NH2, was generated with tyrosine added at position 0. This peptide at 2-20 nM had growth-promoting effects on both normal and malignant human bronchial epithelial cells. Y-23-R-NH2 bound to specific high-affinity receptors (Kd = 2.8 +/- 1.4 x 10(-11) M) present at 1-2 x 10(4) binding sites per cell. Ligand binding was not inhibited by recombinant insulin or recombinant IGF-I. Furthermore, a monoclonal antibody antagonist to the IGF-I receptor (alpha IR3) did not suppress the proliferative response induced by Y-23-R-NH2. In addition, C-terminal amidation was shown to be important in receptor recognition since the free-acid analog of IBE1 (Y-23-R-OH) did not effectively compete for binding and was not a potent agonist of proliferation. Immunoblot analysis of human lung tumor cell line extracts using an antibody raised against Y-23-R-NH2 detected a low molecular mass band of approximately 5 kDa, implying that a protein product is produced that has immunological similarity to IBE1. Extracts of human, mammalian, and avian livers analyzed on an immunoblot with the anti-Y-23-R-NH2 antibody contained proteins of approximately 21 kDa that were specifically recognized by the antiserum and presumably represent an IGF-I precursor molecule. This implies that in species where an IGF-I mRNA with homology to the human IGF-IB E domain has not yet been described, an alternate mRNA must be produced that contains a sequence similar to that of the midportion of the human IGF-IB E domain. Our findings demonstrate that IBE1 is a growth factor that mediates its effect through a specific high-affinity receptor and is most likely conserved in many species.

Pharmacological activities of the MIF-1 analogues Pro-Leu-Gly, Tyr-Pro-Leu-Gly and pareptide

The pharmacological activities of the related free acid analogues of MIF-1, Pro-Leu-Gly (PLG) and Tyr-Pro-Leu-Gly (YPLG), were investigated because of the possibility that they may be formed during the digestion of milk and wheat proteins in vivo. The amino acid sequences-Tyr-Pro-Leu-Gly- and -Pro-Leu-Gly- are present in proteins from these foods. Chronic administration of either PLG (0.25 mg/kg, SC, BID) or the control substance, pareptide (0.25 mg/kg, SC, BID), antagonized the development of tolerance to the cataleptic effects of haloperidol in mice. The effect of YPLG (0.25 mg/kg, SC, BID) on the development of this tolerance was borderline and not statistically significant. Nanomolar concentrations of PLG, YPLG, and pareptide each increased the in vitro binding of 3H-apomorphine to rat striatal receptors. In this in vitro system, bell-shaped dose response curves were observed for each peptide. The effects of these peptides on tolerance development and apomorphine binding are similar to those previously reported for MIF-1 and demonstrate that amidation at the carboxyl terminus is not required for biological activity.

Teratogenesis and reproductive safety evaluation of the retinoid etretin (Ro 10-1670).

Reproduction and teratology studies were performed with etretin, the free acid analog of the retinoid etretinate. The lowest teratogenic dose of etretin in mice, rats and rabbits was 3, 15 and 0.6 mg/kg, respectively. In all three species, the lowest dose which was embryolethal, fetolethal or reduced the survivability of the pups during lactation was 2-3 times higher than the above doses. In rats, the lowest effective dose of etretin was 3 mg/kg in both the study for fertility and general reproductive performance and the peri- and postnatal study. The main adverse effect in these two experiments was a reduced survival of the F1-generation.

Pharmacokinetic studies of amino acid analogues of 2-nitroimidazole, new hypoxic cell radiosensitizers

A series of new analogues of 2-nitroimidazole has been synthesized by inserting various amino acids at 1-position through an amide bond. The ethyl esters of N-α-[(2-nitro-1-imidazolyl)acetyl]-L-phenylalanine and N-α-[2-nitro-1-imidazolyl)acetyl]-L-tyrosine were found to be the most effective radiosensitizers in vitro against hypoxic Chinese hamster (V-79) cells. The sensitizer enhancement ratios (SER) of these derivatives were 2.2 and 2.3 respectively at 1 mM concentration after 2 hr exposure under hypoxia. However, the free acid of phenylalanine analogue was less active as a radiosensitizer and required 5 mM concentration to produce SER of 1.9. In contrast, the free acid of tyrosine analogue was inactive in this test system. The pharmacokinetic studies with the esters revealed their rapid hydrolysis in serum to the corresponding acids within 5 minutes as detected by HPLC. The pharmacokinetic parameters were therefore determined by employing the free acid analogues and solubilizing them as their sodium salts. The drugs were administered intraperitoneally at OS mg/g dose level to C-57 mice bearing B16 melanoma. These agents were cleared from the plasma rapidly with an apparent t sol1 2 of 18.8 and 15.6 min respectively. Peak tumor concentration of approximately 217 ug/g was achieved within 15 min with phenylalanine analogue. The tumor to brain ratio was 10:1 suggesting that this agent is excluded from CNS and that the phenylalanine analogue should be considered a potentially less neurotoxic radiosensitizer than misonidazole.

Corticotropin releasing factor stimulation of protein carboxylmethylation in mouse pituitary tumor cells

A putative role for the protein carboxylmethvlasc (PCM) enzyme has been suggested in exocvtotic secretion. The involvement of 3H-methyl incorporation into protein carboxylmethyl esters during corticotropin releasing factor (CRF)-induced ACTH secretion from AtT-20/D16-16 mouse pituitary cells was investigated. Protein carboxylmethylation and ACTH secretion both increased as a function of extracellular CRF concentration, and both processes were temporally parallel up to 60 min incubation. The less potent [Met(O) 21]-CRF also stimulated increases in protein carboxylmethylation and ACTH secretion. The free acid analogue of CRF did not alter either process. A combination of the PCM inhibitors, 3-deazaadenosine and l-homocysteine thiolactone, reduced both CRF-stimulated protein carboxylmethylation and ACTH release. Dexamethasone, known to inhibit ACTH secretion and synthesis, inhibited both CRF-stimulate protein carboxylmethylation and ACTH secretion.

Corticotropin-releasing factor stimulates phospholipid methylation and corticotropin secretion in mouse pituitary tumor cells.

The 41-residue synthetic ovine corticotropin-releasing factor (CRF; corticoliberin) has been shown to stimulate release of corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin from AtT-20/D16-16 mouse pituitary tumor cells. Phospholipid methylation of phosphatidylethanolamine to phosphatidylcholine with S-adenosylmethionine as methyl donor has been suggested as a possible membrane transduction mechanism for some receptor-induced events. CRF increased phospholipid methylation in pituitary tumor cells at concentrations that also stimulated immunoreactive ACTH secretion, and both processes increased linearly and in parallel with time. The methionine sulfoxide derivative of CRF was less potent than CRF was in stimulating both phospholipid methylation and hormone secretion, and the COOH-terminal free acid analogue of CRF had no effect on either process. CRF-induced increases in phospholipid methylation and ACTH secretion were reduced when cells were treated with the phospholipid methyltransferase inhibitors 3-deazaadenosine and L-homocysteine thiolactone. These CRF-stimulated effects were also blocked by the glucocorticoid dexamethasone. It is suggested that phospholipid methylation may be a CRF receptor-mediated event associated with ACTH release in pituitary tumor cells.

Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone

The synthetic decapeptide “luteinizing hormone-releasing hormone” (LH-RH) was rendered antigenic by reaction of its histidine or tyrosine residues (7 : 3 approx.) with p-diazonium phenylacetic acid and coupling of the azo-derivatives formed to bovine serum albumin (BSA). Immunization of rabbits yielded antisera that bound 125I-labeled LH-RH (approx. 50 pg) at dilutions up to 1:200, 000 and showed no cross-reaction with unrelated hypothalamic and pituitary hormones, extracts from rat cerebral cortex, and with small fragments of LH-RH. Cross-reaction was minimal (0.2%) with the free acid analogue of LH-RH, and moderate with des -pGlu LH-RH (20%), des -pGlu-His-LH-RH (2.4%) and with LH-RH analogues in which a single residue (No. 4–6 or No. 8) was exchanged by an amino-acid of similar character (1.2–12%). Biologically active hypothalamic extract and LH-RH produced parallel 125I-LH-RH-binding inhibition curves, providing immunochemical support for the identity of the native releasing hormone with synthetic LH-RH.