Endogenous morphine modulates acute thermonociception in mice

The endogenous synthesis of morphine has been clearly demonstrated throughout the phylogenesis of the nervous system of mammals and lower animals. Endogenous morphine, serving as either a neurotransmitter or neurohormone, has been demonstrated in the nervous system of both vertebrates and invertebrates. As one of the effects of exogenous morphine is the modulation of pain perception, we investigated the effects that the depletion of endogenous morphine had on nociceptive transmission. The immunoneutralization of endogenous morphine from brain extracellular spaces was obtained through the intracerebroventricular administration of affinity purified anti-morphine IgG to mice, which then underwent the hot plate test. Endogenous morphine immunoneutralization decreased thermal response latency and attenuated the anti-nociceptive effect of the mu selective agonist DAMGO in hot plate test suggesting that endogenous morphine is involved in pain modulation.     

Polymorphonuclear leukocyte-myocyte interaction: an early event in collar-induced rabbit carotid intimal thickening

The importance of mononuclear leukocyte (MO) adhesion to dysfunctional endothelium and migration to the subendothelial space in the early phases of atherogenesis is well established. Few studies have addressed the relevance of polymorphonuclear leukocytes (PMNs) in the context of evolving lesions, and nothing is known about PMN interaction with vascular smooth muscle cells (SMCs). In this study, we investigated leukocyte/SMC interactions in a model of rabbit carotid injury induced by placement of a silastic collar. This procedure leads to the development of intimal thickening characterized by SMC accumulation preceded by an abundant leukocyte infiltration. By transmission electron microscopy and immunocytochemistry, we demonstrated the occurrence of PMN infiltration starting at 6 h and ceasing within 72 h after collar placement. A previously unknown extensive interaction between medial myocytes and PMNs was detected, referring to emperipolesis, an active phenomenon of cells engulfing other cells in a process other than phagocytosis. PMNs, but not MOs, were internalized by SMCs, which showed ultrastructural features intermediate between the true contractile and the fully synthetic phenotype without exhibiting any sign of injury. Emperipolesis preceded any detectable cell proliferation in the vessel wall and disappeared within 72 h, following the kinetic of PMN infiltration in the vessel wall. In summary, our findings show the occurrence of an active and selective interaction between PMNs and SMCs via emperipolesis during the early phases of intimal thickening after perivascular collaring. However, the overall etiology of the phenomena described in the present study and their pathophysiological significance should be further investigated.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Absence of the CD1 molecule up-regulates antitumor activity induced by CpG oligodeoxynucleotides in mice

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene(-/-)/transgenic V(alpha)14/Vbeta8.2 mice that generate NKT cells; J(alpha)281(-/-) mice and CD1(-/-) mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in J(alpha)281(-/-) mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1(-/-) mice the inhibition was dramatic. The increased tumor inhibition in CD1(-/-) correlated with a significantly higher ratio of IFN-gamma-IL-4 production in response to CpG as compared with C57BL/6 and J(alpha)281(-/-) mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-gamma- and IL-4-producing cells revealed that the differences between CD1(-/-) and C57BL/6 in the production of these two cytokines were mainly due to CD3(+) T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of V(alpha)14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.     

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H(2)O(2)) levels in all the internodes, as evidenced in vivo by 3,3'-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H(2)O(2) both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H(2)O(2) production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.     

Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Blend uniformity analysis using stream sampling and near infrared spectroscopy

A near infrared spectroscopic method was developed to determine drug content in a 20% (wt/wt) ibuprofen and spray-dried hydous lactose blend. A blending profile was obtained after blending for 0.5, 1, 3, 5, 10, and 20 minutes. Stream sampling was used to collect about 20 blend samples at each of the blending times from a laboratory scale V-blender. The samples collected were used to develop a near infrared calibration model. The calibration model was then used to determine the drug content of unknown samples from 2 validation blends. The validation blends were not included in the calibration model; they were used to evaluate the effectiveness of the calibration model. A total of 45 samples from the 2 validation blends were predicted by the near infrared calibration model and then analyzed by a validated UV spectrophotometric method. The root mean square error of prediction for the first validation blend was 5.69 mg/g and 3.30 mg/g for the samples from the second blend. A paired t test at the 95% confidence level did not indicate any differences between the drug content predicted by the near infrared spectroscopy (NIRS) method and the validated UV method for the 2 blends. The results show that the NIRS method could be developed while the blending profile is generated and used to thoroughly characterize a new formulation during development by analyzing a large number of samples. The new formulation could be transferred to a manufacturing plant with an NIRS method to facilitate blend uniformity analysis.