The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.     

DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50k bp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 microM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin.     

Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: a comparison with other osmotic agents and free radical scavengers

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na(v) channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na(v) channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I(Na) amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na(v) channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na(v) channel subtype, possibly to alter or reduce toxin association.     

Cellular uptake of a catechol iron chelator and chloroquine into Plasmodium falciparum infected erythrocytes

Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained after 2 hr of exposure. After 2 hr exposure, it reaches intracellular levels that are 4- to 10-fold higher in infected red blood cells than those attained in normal erythrocytes. There is quite a good correlation between the accumulation of chloroquine and FR160 in the different strains (r=0.939) and in the IC(50) values (r=0.719). In contrast, the accumulation of FR160 and its activity is poorly correlated (r=0.500), suggesting that activity of FR160 may be independent of its penetration into infected erythrocytes. The mechanism of accumulation is yet unknown but based on inhibitor studies, the uptake of FR160 seems to be not associated with the calcium pump or channel, the potassium channel or the Na(+)/H(+) exchanger. Combinations of FR160 with verapamil, diltiazem, clotrimazole, amiloride, diazoxide, 4-aminopyridine, and picrotoxin should be avoided (antagonistic effects). The potent in vitro activity of FR160 on chloroquine-resistant strains or isolates, its lower toxicity against Vero cells, its mechanisms of action, its capacity to reach rapidly and accumulate into infected erythrocytes suggest that FR160 holds much promise as a new structural lead and effective antimalarial agent or at least a promising adjuvant in treatment of malaria.     

Characterization of the molecular interactions of interleukin-8 (CXCL8), growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) with the human CXCR2

Neutrophil recruitment to inflammatory sites is mediated by two related receptors: CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2). Both receptors share two ligands, interleukin-8 (CXCL8) and GCP-2 (CXCL6), whereas several chemokines, including growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) are specific for CXCR2. The objective of this study was to map the different amino acids involved in the binding and activation/inhibition of human CXCR2. This was performed by exchanging non-conserved amino acids of CXCR2 with their counterparts in CXCR1. The mutants generated showed that: (a) for CXCL8 binding, the N-terminus of CXCR1 and the second extra-cellular loop of CXCR2 are determinant, the N-terminus of CXCR2 is not sufficient and the transmembrane domain seven is probably involved; (b) for CXCL1, the N-terminus of CXCR2 is necessary but not sufficient for binding. The activation study indicated that amino acids critical for activation are not necessarily involved in binding process. Finally, the mechanism of binding of a non-peptide antagonist on CXCR2 was investigated: it occurred through epitopes (a) which were disseminated within the receptor, (b) which differed according to the use of CXCL8 or CXCL1 as a competitor and (c) which did not necessarily overlap with agonist binding sites. We also showed that inhibition of binding and inhibition of activation involved different amino acids.     

Recombinant arginine deiminase as a differential modulator of inducible (iNOS) and endothelial (eNOS) nitric oxide synthetase activity in cultured endothelial cells

Modulation of the extracellular level of arginine, substrate for nitric oxide synthetases, is a promising modality to alleviate certain pathological conditions where excess nitric oxide (NO) is produced. However, complications arise, as only preferential inhibition of the inducible nitric oxide synthetase (iNOS), but not endothelial nitric oxide synthetase (eNOS), is desired for the treatment of NO over-production. We investigated the effect of arginine deprivation mediated by a recombinant arginine deiminase (rADI) on the activity of iNOS and eNOS in an endothelial cell line, TR-BBB. Our results demonstrated that cytokine-induced NO production depends on the extracellular arginine as substrate. However, if sufficient citrulline is present in the medium, A23187-activated NO production by eNOS does not rely on extracellular arginine. Treatment with rADI can markedly inhibit cytokine-induced NO production via iNOS, but not A23187-activated NO production via eNOS. Our results also showed that the decrease of NO production by iNOS could be achieved by depleting arginine from the medium even under the conditions that would up-regulate iNOS expression. Thus, rADI appears to be a novel selective modulator of iNOS activity that may be a used as a tool in the study of pathological disorders where NO over-production plays a key role.     

Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea,quantitated using the single cell gel electrophoresis (Comet) assay

Enhanced DNA repair activity is important for the development of cellular resistance to alkylating agents. Here, we quantitated the kinetics of DNA excision repairs initiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in human leukemia CCRF-CEM cells. CEM cells that had been established resistant to BCNU (CEM-R) were evaluated in comparison with parental CEM cells (CEM-S). The excision repair kinetics were quantitated as the amount of DNA single strand breaks, which were generated from the incision/excision of the damaged DNA and were diminished by the rejoining of renewed DNA, using the single cell gel electrophoresis (Comet) assay. CEM-R cells were 10-fold more resistant to BCNU than CEM-S cells, and also showed cross-resistance to melphalan and cisplatin. In response to the treatment with BCNU, both CEM-S and CEM-R cells initiated an incision/excision reaction at the end of the incubation period, and completed the rejoining process within 4 hr. While CEM-S cells could not repair the damage induced by the high concentration of BCNU, CEM-R cells completed the repair process regardless of BCNU concentrations, suggesting enhanced excision repairs in CEM-R cells. The excision repair activity of CEM-R cells was increased with regard to the incision reaction and to the rate of the repair. Similar results were obtained using ultraviolet C, suggesting enhanced nucleotide excision repair in CEM-R cells. Thus, the enhanced DNA excision repairs were successfully quantitated in the resistant leukemic cell line using the Comet assay. The evaluation of the repair activity may predict the sensitivity of cancer cells to chemotherapy and provide a clue to overcome the resistance.     

Accessibility of endothelial and inducible nitric oxide synthase to the intracellular citrulline-arginine regeneration pathway

This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine (L-arg) regeneration from citrulline (L-cit), plays a pivotal role in supplying L-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood-brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of the L-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without alpha-methyl-DL-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown in L-arg-free/L-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only in L-arg containing medium, but also L-arg-free and L-arg-free/L-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed in L-arg containing medium, not in L-arg-free or L-arg-free/L-cit-supplemented medium, and it was not inhibited by MDLA in the L-arg containing medium. Our results indicate that extracellular L-arg was the only L-arg pool for cytokine-induced NO production and intracellular L-arg regenerated from L-cit via AS pathway was the major L-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.     

Three weeks release BCNU loaded hydrophilic-PLGA microspheres for interstitial chemotherapy: Development and activity against human glioblastoma cells

The aim of this study is the development of microspheres of BCNU for intracranial administration, as an alternative to marketed novel Gliadel® Implant in the treatment of brain tumours. H poly-lactide-co-glycolide biodegradable microspheres of BCNU with a mean size of 33.5 ± 1.8 µm were obtained by an oil-in-water emulsion solvent evaporation method. Their small size would allow their intracranial administration through a needle by cerebral stereotaxia if tumour recurrence occurs, without a surgical intervention, as Gliadel® needs. BCNU was released from these microspheres during 21 days, mainly by a mechanism of diffusion from the polymer matrix (K = 2.91 mg days^?½). The cytotoxic effects of these microspheres on human glioblastoma cells were demonstrated all through 21 days and the value of percentage of viable cells was less than 40%. These microspheres should be commercialized as a freeze-dried product to keep at ?20°C. Three hundred and twenty milligrams of microspheres contain 61.6 mg of BCNU, the same amount of BCNU contained in 1600 mg or eight wafers of Gliadel usually implanted after the tumour resection.     

Synergism between staurosporine and drugs inducing endoplasmic reticulum stress

Drugs causing endoplasmic reticulum or mitochondrial dysfunction may trigger apoptosis in eukaryotic cells. The thiol reagent dithiothreitol (DTT) belongs to the first group whereas the protein kinases inhibitor staurosporine acts on mitochondria. Since the endoplasmic reticulum and the mitochondrial pathways of apoptosis may converge in common steps, we examined the possibility of synergism between these two drugs. Using the activation of caspase-3 as indicator of apoptosis, we found that in two cell lines, Jurkat and Mono-Mac 6, staurosporine and DTT elicited apoptosis with a different pattern: staurosporine acted rapidly and at nanomolar concentrations while DTT acted slowly and at higher concentrations (1mM). When staurosporine and DTT were combined, the proapoptotic action was increased. This was confirmed examining late apoptotic events such as the translocation of phosphatidylserine across the plasma membrane and the cleavage of the antiapoptotic protein Mcl-1. The use of subthreshold DTT concentrations and isobologram analysis demonstrated the synergic nature of the interaction. Tunicamycin, a drug that, like DTT, inhibits protein folding in the endoplasmic reticulum also increased the proapoptotic effect of staurosporine. In agreement with the interplay between the mitochondrial and the endoplasmic reticulum pathways it was found that both staurosporine and DTT induced cytochrome c release. Furthermore, 90min incubation with DTT did not induce caspase-4 activation while staurosporine alone or in combination with DTT stimulated caspase-4 activity. We conclude that staurosporine is more active in cells undergoing endoplasmic reticulum stress. This synergism may warrant evaluation to establish whether the anticancer activity of staurosporine is also enhanced.     

The effects of natural ligands of hormone receptors and their antagonists on telomerase activity in the androgen sensitive prostatic cancer cell line LNCaP

The effects of the 17beta-estradiol, dihydrotestosterone and hormone antagonists tamoxifen and bicalutamide on telomerase activity and expression of cell cycle related proteins in the androgen-sensitive prostatic cancer cell line LNCaP were studied. The cell line was grown in RPMI supplemented with 2.5% charcoal-stripped FBS for 72 hr. The IC(50) of tamoxifen and bicalutamide and the optimal stimulatory concentrations of 17beta-estradiol and dihydrotestosterone were determined by means of the cell-viability assay, the activity of telomerase was measured by the telomere repeat amplification protocol (TRAP) and the expression of proteins was analysed by the Western blot technique. 17beta-estradiol stimulated cell growth more effectively than dihydrotestosterone whereas hormone antagonists tamoxifen and bicalutamide caused a significant decrease in cell viability. The treatment of cells by a combination of low doses of 17 beta-estradiol and dihydrotestosterone stimulated cells stronger than treatment by a single hormone. Only 17beta-estradiol, in concentration of 10nM, increased strongly the expression of p21(Waf1/Cip1) and increased slightly telomerase activity in the LNCaP cells. 50 microM of bicalutamide down-regulated the levels of the androgen receptor, the proliferating cell nuclear antigen and telomerase activity, and up-regulated the expression of p27(Kip1). We hereby describe the first observation of the influence of bicalutamide on telomerase activity and a positive correlation between the effect of 17beta-estradiol and the induction of both the endogenous cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), and telomerase activity in a prostatic cancer cell line LNCaP. These findings can shed a new light on the steroid-signaling pathway in prostate cancer cells.     

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.     

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.     

Preliminary Assessment of the Immune Response to Olea europaea Pollen Extracts Encapsulated into PLGA Microspheres

In this study, poly (d,l-lactide-co-glycolide) (PLGA) microspheres encapsulating Olea europaea pollen extracts were prepared by using the double emulsion (w/o/w) based on a solvent evaporation/extraction method. The resulting microspheres were 1.93 μm in size. The total allergen loading and surface-associated allergen were 8 and 0.64%, respectively. The release of the allergen from the microspheres showed a biphasic profile with an initial burst release followed by a sustained release phase. Finally, the polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the encapsulation process does not affect the stability of the protein. We describe here some preliminary observations concerning the use of these microspheres as parenteral antigen delivery systems for immunization with O. europaea pollen extracts, in a small animal model, the mouse.     

Evaluation of the degradation of clonidine-loaded PLGA microspheres

Abstract

Context: The release of an encapsulated drug is dependent on diffusion and/or degradation/erosion processes.

Objective: This work aimed to better understand the degradation mechanism of clonidine-loaded microparticles.

Methods: Gel permeation chromatography was used to evaluate the degradation of the polymer. The water-uptake and the weight loss were determined gravimetrically. The swelling behaviour and the morphological changes of the formulations were observed by microscopy. The glass transition temperature and the crystallinity were also determined by differential scanning calorimetry and X-ray diffraction, respectively. The pH of the medium and inside the microspheres was assessed.

Results: The microspheres captured a large amount of water, allowing a decrease in the molecular weight of the polymer. The pH of the medium decreased after release of the degradation products and the pH inside the microparticles remained constant due to the neutralization of these acidic products.

Conclusion: Clonidine and buffers both had an action on the degradation.     

Preparation and characterization of D,L-PLA loaded 17-β-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-β-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718–880 nm (inert micro-particles) and 3–4 µm (drug loaded microparticles). The encapsulation efficiency was ～80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-β-estradiol.     

Measurement of Protein Diffusion Through Poly(d,l-Lactide-Co-Glycolide)

A novel method was developed for studying the diffusion of proteins through poly(d,l-lactide-co-glycolide) (PLG), using a diffusion cell. To develop improved formulations for the controlled release of encapsulated drugs it is important to understand the underlying release mechanisms. When using low-molecular-weight PLG as the release-controlling polymer, diffusion through the pores is often proposed as the main release mechanism. The experimental set-up and method of determining the diffusion coefficient were thoroughly evaluated with regard to the reliability and the influence of the stirring rate. A procedure for spraying thin films of PLG onto a filter, which could be placed in the diffusion cell, was optimized. The method was then applied to the determination of the diffusion coefficient of human growth hormone (hGH) through a PLG film. The results show that the method enables measurements of the diffusion coefficient through the polymer film. Neither the stirring rate nor the concentration of hGH influenced the diffusion coefficient. The diffusion coefficient of hGH through degraded PLG films was 5.0 · 10^{? 13} m²/s, which is in the range that could be expected, i.e., several orders of magnitude smaller than its the diffusivity in pure water. The reproducibility was good, considering the dynamic properties of PLG, i.e., the difference in diffusion coefficients, at, for example, different stages of degradation and for different compositions of PLG, is expected to be much higher. The variation is probably also present in PLG films used for controlled-release formulations. Although the PLG film contains a large amount of water, a considerable time elapsed before pores of sufficient size formed and diffusion through the film started. In two-component diffusion experiments, the difference in diffusion rate did not correspond to the difference in molecular weight of the solutes, indicating a size exclusion effect. This method can be used to study the effect of changes in the formulation specification. By studying the change in the diffusion coefficient through the degradation process of PLG, or similar polymers, a better understanding of diffusion and, thus, also release mechanisms can be obtained.     

Agmatine exerts anticonvulsant effect in mice: modulation by alpha 2-adrenoceptors and nitric oxide

The effect of agmatine, an endogenous polyamine metabolite, on seizure susceptibility was investigated in mice. Acute intraperitoneal administration of agmatine (5, 10, 20, 40 mg/kg) had a significant and dose-dependent inhibitory effect on pentylenetetrazole (PTZ)-induced seizures. The peak of this anticonvulsant effect was 45 min after agmatine administration. We further investigated the possible involvement of the alpha(2)-adrenoceptors and L-arginine/NO pathway in this effect of agmatine. The alpha(2)-adrenoceptor antagonist, yohimbine (0.5-2 mg/kg), induced a dose-dependent blockade of the anticonvulsant effect of agmatine. The nitric oxide synthase (NOS) substrate, L-arginine (60 mg/kg), inhibited the anticonvulsant property of agmatine and this effect was significantly reversed by NOS inhibitor N(G)-nitro-L-arginine (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for L-arginine effect. We further examined a possible additive effect between agmatine (1 or 5 mg/kg) and L-NAME (10 mg/kg). The combination of L-NAME (10 mg/kg) with agmatine (5 but not 1 mg/kg) induced a significantly higher level of seizure protection as compared with each drug alone. Moreover, a combination of lower doses of yohimbine (0.5 mg/kg) and L-arginine (30 mg/kg) also significantly decreased the anticonvulsant effect of agmatine. In conclusion, the present data suggest that agmatine may be of potential use in seizure treatment.     

Structures of the glycine-rich diastereomeric peptides bombinin H2 and H4

Skin secretions of the European frog Bombina variegata contain a family of hydrophobic peptides, called bombinins H, which probably play a role in the defense against microbes. These peptides are rich in glycine (25%), which may allow structural polymorphism. Indeed, there is increasing evidence that bombinin H can, dependent on the environment, adopt different conformations. Moreover, some of these peptides contain a d-amino acid; the bombinins H2 and H4 differ from each other in that they contain either L-Ile or D-allo-Ile at position 2.In this paper we report the solution structure obtained by using NMR techniques of the mainly helical conformation, which these peptides adopt upon binding to the bilayer mimetics SDS and DPC. A glycine ridge is exposed at one side of the helix and may provide a helix-helix interaction site. In this respect, the structure of bombinin H resembles the influenza hemagglutinin fusion peptide and the helical conformer of Alzheimer peptide Aβ(25-40). Neither structure nor orientation of bombinin H are affected by the chiral inversion.Environmental conditions can trigger self-aggregation of bombinin H in solution due to hydrophobic interaction. Under these conditions the stereochemistry of the randomly ordered N-terminal segment modulates the preference to fold into a particular conformation.     

Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines

Curcumin, a well-known chemopreventive agent, has been shown to suppress the proliferation of a wide variety of tumor cells through a mechanism that is not fully understood. Cyclin E, a proto-oncogene that is overexpressed in many human cancers, mediates the G(1) to S transition, is a potential target of curcumin. We demonstrate in this report a dose- and time-dependent down-regulation of expression of cyclin E by curcumin that correlates with the decrease in the proliferation of human prostate and breast cancer cells. The suppression of cyclin E expression was not cell type dependent as down-regulation occurred in estrogen-positive and -negative breast cancer cells, androgen-dependent and -independent prostate cancer cells, leukemia and lymphoma cells, head and neck carcinoma cells, and lung cancer cells. Curcumin-induced down-regulation of cyclin E was reversed by proteasome inhibitors, lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, suggesting the role of ubiquitin-dependent proteasomal pathway. We found that curcumin enhanced the expression of tumor cyclin-dependent kinase (CDK) inhibitors p21 and p27 as well as tumor suppressor protein p53 but suppressed the expression of retinoblastoma protein. Curcumin also induced the accumulation of the cells in G1 phase of the cell cycle. Overall, our results suggest that proteasome-mediated down-regulation of cyclin E and up-regulation of CDK inhibitors may contribute to the antiproliferative effects of curcumin against various tumors.