New Metabolic Influencer on Oxytocin Release: The Ghrelin.

BACKGROUND: The hypothalamic⁻pituitary axis by secreting neuropeptides plays a key role in metabolic homeostasis. In light of the metabolic regulation, oxytocin is a potential neuropeptide for therapies against obesity and related disorders. The aim of our study is to measure ghrelin-induced oxytocin secretion in rats and to detect the changes after administration of ghrelin antagonist. METHODS: Ghrelin was administrated centrally (intracerebroventricular, i.c.v., 1.0, 10.0, and 100.0 pmol) or systemically (intravenous, i.v., 1.0, and 10.0 nmol). [d-Lys³]-GHRP-6 ghrelin antagonist was injected 15 min before ghrelin injection in a dose of 10.0 pmol i.c.v. and 10.0 nmol i.v. RESULTS: Either i.c.v. or i.v. administration of ghrelin dose-dependently increased the plasma oxytocin concentration. Following pretreatment with the ghrelin antagonist [d-Lys³]-GHRP-6, the high plasma oxytocin level induced by ghrelin was significantly reduced. CONCLUSION: The results indicate that the release of oxytocin is influenced directly by the ghrelin system. Examination of the mechanism of ghrelin-induced oxytocin secretion is a new horizon for potential therapeutic options.

Pharmacological Modulation of Endogenous Opioid Activity to Attenuate Neuropathic Pain in Rats.

We showed previously that spinal metabotropic glutamate receptor 1 (mGluR1) signaling suppresses or facilitates (depending on the stage of estrous cycle) analgesic responsiveness to intrathecal endomorphin 2, a highly mu-opioid receptor-selective endogenous opioid. Spinal endomorphin 2 antinociception is suppressed during diestrus by mGluR1 when it is activated by membrane estrogen receptor alpha (mERα) and is facilitated during proestrus when mGluR1 is activated by glutamate. In the current study, we tested the hypothesis that in female rats subjected to spinal nerve ligation (SNL), the inhibition of spinal estrogen synthesis or blockade of spinal mERα/mGluR1 would be antiallodynic during diestrus, whereas during proestrus, mGluR1 blockade would worsen the mechanical allodynia. As postulated, following SNL, aromatase inhibition or mERα/mGluR1 blockade during diestrus markedly lessened the mechanical allodynia. This was observed only on the paw ipsilateral to SNL and was eliminated by naloxone, implicating endogenous opioid mediation. In contrast, during proestrus, mGluR1 blockade worsened the SNL-induced mechanical allodynia of the ipsilateral paw. Findings suggest menstrual cycle stage-specific drug targets for and the putative clinical utility of harnessing endogenous opioids for chronic pain management in women, as well as the value of, if not the necessity for, considering menstrual cycle stage in clinical trials thereof. PERSPECTIVE: Intrathecal treatments that enhance spinal endomorphin 2 analgesic responsiveness under basal conditions lessen mechanical allodynia in a chronic pain model. Findings provide a foundation for developing drugs that harness endogenous opioid antinociception for chronic pain relief, lessening the need for exogenous opioids and thus prescription opioid abuse.

Degarelix versus luteinizing hormone-releasing hormone agonists for the treatment of prostate cancer.

INTRODUCTION: Androgen deprivation therapy (ADT) is the mainstay for advanced, hormone-sensitive prostate cancer, and options include surgical castration, luteinizing hormone-releasing hormone (LHRH) agonist, and more recently, gonadotropin releasing hormone (GnRH) antagonist therapy. Our understanding of the mechanisms and adverse effects of ADT has increased substantially, including the class-specific adverse effects of ADT. Areas covered: This review will summarize the pharmacodynamic and pharmacokinetic properties of the GnRH antagonist degarelix and its role in the management of advanced prostate cancer, the clinical evidence supporting its regulatory approval, as well as potential benefits and disadvantages over traditional LHRH agonist therapy. Expert opinion: Degarelix represents a newer class of ADT that results in a rapid and reliable decline in serum testosterone, a quality that makes it particularly advantageous in men presenting with symptomatic, hormone-sensitive prostate cancer. Due to differences in mechanism of action, there is observational data suggesting a potential cardiovascular and even oncologic benefit over traditional LHRH agonist therapy. Further research is ongoing to more clearly define this potential benefit.

Short Exogenous Peptides Regulate Expression of CLE, KNOX1, and GRF Family Genes in Nicotiana tabacum.

Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10-7-10-9 M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants.

BACKGROUND: Fire blight is an important disease affecting rosaceous plants. The causal agent is the bacteria Erwinia amylovora which is poorly controlled with the use of conventional bactericides and biopesticides. Antimicrobial peptides (AMPs) have been proposed as a new compounds suitable for plant disease control. BP100, a synthetic linear undecapeptide (KKLFKKILKYL-NH2), has been reported to be effective against E. amylovora infections. Moreover, BP100 showed bacteriolytic activity, moderate susceptibility to protease degradation and low toxicity. However, the peptide concentration required for an effective control of infections in planta is too high due to some inactivation by tissue components. This is a limitation beause of the high cost of synthesis of this compound. We expected that the combination of BP100 with lysozyme may produce a synergistic effect, enhancing its activity and reducing the effective concentration needed for fire blight control. RESULTS: The combination of a synhetic multifunctional undecapeptide (BP100) with lysozyme produces a synergistic effect. We showed a significant increase of the antimicrobial activity against E. amylovora that was associated to the increase of cell membrane damage and to the reduction of cell metabolism. Combination of BP100 with lysozyme reduced the time required to achieve cell death and the minimal inhibitory concentration (MIC), and increased the activity of BP100 in the presence of leaf extracts even when the peptide was applied at low doses. The results obtained in vitro were confirmed in leaf infection bioassays. CONCLUSIONS: The combination of BP100 with lysozyme showed synergism on the bactericidal activity against E. amylovora and provide the basis for developing better formulations of antibacterial peptides for plant protection.

Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats.

Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague-Dawley rats were injected with TAC (0.2 mg/kg/day) for 4-8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2 ± 30.1 mg/24 h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9 ± 22.5 mg/24 h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury.

Novel endomorphin-1 analogs with C-terminal oligoarginine-conjugation display systemic antinociceptive activity with less gastrointestinal side effects.

In recent study, in order to improve the bioavailability of endomorphin-1 (EM-1), we designed and synthesized a series of novel EM-1 analogs by replacement of L-Pro(2) by ß-Pro, D-Ala or Sar, together with C-terminal oligoarginine-conjugation. Our results indicated that the introduction of D-Ala and ß-Pro in position 2, along with oligoarginine-conjugation, didn't significantly decrease the µ-affinity and in vitro bioactivity, and the enhancement of arginine residues did not markedly influence the µ-affinity of these analogs. All analogs displayed a significant enhancement of stability, which may be due to increased resistance to proline-specific enzymatic degradation. Moreover, following intracerebroventricular (i.c.v.) administration, analogs 1, 2, 4 and 5 produced significant antinociception and increased duration of action, with the ED50 values being about 1.8- to 4.2-fold less potent than that of EM-1. In addition, our results indicated that no significant antinociceptive activity of EM-1 was seen following subcutaneous (s.c.) injection, whereas analogs 1, 2, 4 and 5 with equimolar dose induced significant and prolonged antinociception by an opioid and central mechanism. Herein, we further examined the gastrointestinal transit and colonic propulsive latencies of EM-1 and its four analogs administered centrally and peripherally. I.c.v. administration of EM-1 and analogs 1, 2, 4 and 5 significantly delayed gastrointestinal transit and colonic bead propulsion in mice, but the inhibitory effects induced by these analogs were largely attenuated. It is noteworthy that no significant gastrointestinal side effects induced by these four analogs were observed after s.c. administration. Our results demonstrated that combined modifications of EM-1 with unnatural amino acid substitutions and oligoarginine-conjugation gave an efficient strategy to improve the analgesic profile of EM-1 analogs but with less gastrointestinal side effects when administered peripherally.

Heat shock protein 90 inhibitor regulates necroptotic cell death via down-regulation of receptor interacting proteins.

17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) acts as an inhibitor of heat shock protein 90 (HSP 90), which serves as a nodal protein of diverse signaling networks leading to a variety of biological implications. HSP90 plays the role of a chaperone for a variety of client proteins including receptor interacting protein 1 (RIP1). Since RIP1 and RIP3 are, respectively, required for zVAD- and tumor necrosis factor alpha (TNFα)-mediated necrotic cell death, we pursued to address the effects of DMAG on receptor-and nonreceptor-mediated necroptotic cell death. DMAG facilitated the degradation of receptor interacting protein 3 (RIP3) as well as RIP1, a known client protein of HSP90, in L929 cells. Consequently, DMAG rendered cells more sensitive to TNFα stimulation while it rescued cells from necrotic cell death caused by zVAD. From this study, we propose that DMAG-downregulated RIP1 can shift cell death typing from necroptosis to apoptosis. In contrast, the protective effect of DMAG on zVAD-induced cytotoxicity could be partly explained by the fact that zVAD mediates cytotoxicity via a RIP1 -dependent route. In summary, functional disruption of HSP90 by DMAG destabilized necroptosis proteins RIP1 and RIP3, which in turn regulated zVAD- and TNFα-induced necroptosis. Therefore, pharmacological modulation of necroptotic cell death through HSP90 could be a promising strategy for overcoming cancer drug resistance or protecting ischemic cell death.

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications.

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant's biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO3 or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO3-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.

Structure-activity relationships of sugar-based peptidomimetics as modulators of amyloid ß-peptide early oligomerization and fibrillization.

Alzheimer's disease is a neurodegenerative disorder linked to oligomerization and fibrillization of amyloid ß peptides. Aß1-42 being the most aggregative and neurotoxic amyloid peptide, we report herein the capacity of sugar-based pentapeptide analogs to modulate the Aß1-42 aggregation process using thioflavin fluorescence and transmission electron microscopy assays. The importance of the free hydroxyl groups of the sugar moiety, used as a ß-breaker element, is confirmed since hydroxylated compounds inhibit the aggregation process while benzylated ones enhance it. Furthermore, the most effective molecules were also evaluated by a recently developed capillary electrophoresis method, providing in vitro monitoring of the crucial, very early stages of the self-assembly process. This technique allowed us to investigate the effect of these compounds on the small non-fibrillar Aß1-42 oligomers suspected by several groups worldwide as highly neurotoxic. We clearly demonstrated that molecules delaying the aggregation can stabilize the monomeric peptide or promote the formation of soluble oligomeric species. In contrast, molecules that accelerate the aggregation can prevent the presence of small toxic oligomers.

Replacement of bortezomib with carfilzomib for multiple myeloma patients progressing from bortezomib combination therapy.

In this open-label, intra-patient phase I/II trial, bortezomib was replaced with carfilzomib (escalated from 20 to 45 mg/m(2) on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle) for multiple myeloma (MM) patients who progressed while on or within 12 weeks of receiving a bortezomib-containing combination regimen. Study objectives included determination of the maximum tolerated dose (MTD), overall response rate (ORR), clinical benefit rate (CBR), time to progression, time to response, duration of response, progression-free survival and overall survival (OS). Of 38 registered patients, 37 were treated and evaluable for efficacy and safety. Thirty-one carfilzomib-based regimens using 14 different drug combinations were tested. One regimen (carfilzomib (45 mg/m(2)), ascorbic acid (1000 mg) and cyclophosphamide (2.2 mg/kg)) reached MTD. ORR and CBR were 43.2 and 62.2%, respectively. Median progression-free survival, time to progression and OS were 8.3, 9.9 and 15.8 months, respectively. Hematologic adverse events (AEs; ⩾grade 3) included lymphopenia (35.1%), thrombocytopenia (24.3%), anemia (10.8%) and neutropenia (10.8%). Nonhematologic AEs (⩾grade 3) included fever (5.4%) and hypokalemia (5.4%). These results demonstrate that replacing bortezomib with carfilzomib is safe and can be effective for MM patients failing bortezomib-containing combination regimens. This trial was registered at http://www.clinicaltrials.gov (#NCT01365559).

Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats.

Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N(G)-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5-2.5 µg) and endomorphin-1 (2.5-20 µg) in acute pain and morphine (10-40 µg) and endomorphin-1 (5-20 µg) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED50 for endomorphin-1 (7.1 µg) was higher than that of morphine (1.3 µg) in the tail-flick test. For neuropathic pain, the ED50 value for morphine was much higher (43.2 µg) than that of endomorphin-1 (9.2 µg) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by L-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the effects of endomorphin-1, an opioid ligand with a peptidergic structure.

99mTc-(Me)FGCDEVD, a potential tracer for apoptosis detection.

(Me)FGC(Bz)DEVD was radiolabeled with technetium-99m in high yield. This tracer was preferentially accumulated in apoptotic cells in the in vitro studies. Tumor uptake occurred in vivo after cisplatin injection due to the apoptosis induction, which not observed in the untreated tumors. Therefore, (99m)Tc-(Me)FGCDEVD is a potential tracer for apoptosis detection.

The proteinase/proteinase-activated receptor-2/transient receptor potential vanilloid-1 cascade impacts pancreatic pain in mice.

AIMS: Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice. MAIN METHODS: Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonists into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry. KEY FINDINGS: The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH(2), a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH(2) was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate. SIGNIFICANCE: Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice.

beta-Sheet aggregation of kisspeptin-10 is stimulated by heparin but inhibited by amphiphiles.

The murine 10-residue neurohormone kisspeptin (YNWNSFGLRY) is an important regulator of reproductive behavior and gonadotrophin secretion. It is known to form a random coil in solution, but undergoes a structural change in the presence of membranes although the nature of this change is not fully determined. The peptide's conformational versatility raises the question whether it is also able to form ordered aggregates under physiological conditions, which might be relevant as a storage mechanism. Here we show that heparin induces kisspeptin to form beta-sheet rich amyloid aggregates both at neutral (pH 7.0) and slightly acidic (pH 5.2) conditions. Addition of heparin leads to aggregation after a certain lag phase, irrespective of the time of addition of heparin, indicating that heparin is needed to facilitate the formation of fibrillation nuclei. Aggregation is completely inhibited by submicellar concentrations of zwitterionic and anionic surfactants. Unlike previous reports, our NMR data do not indicate persistent structure in the presence of zwitterionic surfactant micelles. Thus kisspeptin can aggregate under physiologically relevant conditions provided heparin is present, but the process is highly sensitive to the presence of amphiphiles, highlighting the very dynamic nature of the peptide conformation and suggesting that kisspeptin aggregation is a biologically regulatable process.

Intratumoral spatial distribution of hypoxia and angiogenesis assessed by 18F-FAZA and 125I-Gluco-RGD autoradiography.

UNLABELLED: The hypoxia-inducible factor-1 alpha (HIF-1 alpha) activates angiogenesis in response to cellular hypoxia, suggesting a spatial correlation between angiogenesis and tissue hypoxia. METHODS: Using digital autoradiography of coinjected 18F-labeled azomycin arabinoside (8F-FAZA) (assessing regional hypoxia) and a glycosylated RGD-containing peptide (125I-3-iodo-dTyr(4)-cyclo(-Arg-Gly-Asp-dTyr-Lys(SAA)-), or 125I-Gluco-RGD) (assessing angiogenesis via binding to alpha v beta 3 integrin receptors on endothelial cells) performed on 22 EMT6 tumor xenografts, we investigated the intratumoral spatial distribution of these tracers. We applied a Bayesian bivariate image analysis using the mean tumor-to-muscle ratio as a discriminator, resulting in 4 groups: FAZA high/RGD high (Q1), FAZA low/RGD high (Q2), FAZA low/RGD low (Q3), and FAZA high/RGD low (Q4). In an additional 18 xenografts, the immunohistochemically derived HIF-1 alpha protein distribution was compared with 18F-FAZA autoradiography. Animals were divided into groups breathing either room air or carbogen (95% oxygen, 5% CO2) for 4 h until sacrifice. RESULTS: Under room air conditions, roughly 60% of the tumor surface displayed a spatial coupling of 18F-FAZA and 125I-Gluco-RGD uptake: either high (Q1) or low (Q3) uptake for both tracers, with Q1 indicating spatial association of hypoxia and angiogenesis and Q3 indicating adequate oxygenation without active angiogenesis. However, the remaining approximately 40% of the tumor surface showed discordant 18F-FAZA and 125I-Gluco-RGD uptake, indicating that hypoxia and angiogenesis are not necessarily spatially linked to each other and highlighting substantial intratumoral heterogeneity of the 18F-FAZA and 125I-Gluco-RGD uptake. Although carbogen breathing conditions significantly decreased the mean 18F-FAZA tumor-to-muscle ratio, no significant changes were observed for 125I-Gluco-RGD, indicating that an acute increase in tumor oxygenation did not influence alpha v beta 3 integrin receptor expression. The HIF-1 alpha-positive (HIFpos) tumor cell fraction was not significantly influenced by breathing conditions and covered between 0% and 35% of the total tumor section surface. However, the HIFpos tumor section surface was much smaller than the tumor section surface of increased 18F-FAZA uptake, suggesting that both markers are identifying distinctly different biologic processes associated with hypoxia. CONCLUSION: The study revealed a substantial spatial discordance of the 18F-FAZA and 125I-Gluco-RGD tumor distribution suggesting that hypoxia and angiogenesis are not necessarily spatially linked in malignancies. These results may prove essential in developing advanced targeted systemic chemotherapeutic approaches (such as combinations of hypoxia-activated cytotoxins and antiangiogenic drugs) for hypoxic tumors.

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli.

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.

Effects of esomeprazole on the pharmacokinetics of atazanavir and fosamprenavir in a patient with human immunodeficiency virus infection.

The effects of proton pump inhibitors on the pharmacokinetics of atazanavir and amprenavir (administered as fosamprenavir) were rigorously evaluated in healthy volunteers in two studies, but formal studies in persons infected with human immunodeficiency virus (HIV) are lacking. We describe a 65-year-old man with HIV who underwent a 12-hour intensive pharmacokinetic study while receiving esomeprazole with atazanavir-ritonavir and subsequently, an 8-hour study while receiving esomeprazole with fosamprenavir-ritonavir. Consistent with the data in healthy volunteers, a major interaction between esomeprazole and atazanavir-ritonavir was observed in this patient-marked reductions in atazanavir trough plasma concentration and in the area under the concentration-time curve from 0-24 hours-whereas an interaction between esomeprazole and fosamprenavir-ritonavir was not apparent in this patient.

Minocycline treatment attenuates microglia activation and non-angiotensin II [125I] CGP42112 binding in brainstem following nodose ganglionectomy.

We have previously shown that following unilateral nodose ganglionectomy, [125I] CGP42112 binds to a non-angiotensin II (Ang II) related binding site in rat dorsal motor nucleus of the vagus nerve, ambiguus nucleus and nucleus of the solitary tract. Furthermore, this up-regulated binding site localizes with activated microglia. Given that some tetracyclines may inhibit microglia activation in brain, we examined the effect of minocycline treatment on the binding of [125I] CGP42112 and [3H] PK11195 (an established radioligand for microglia), as well as OX-42 immunoreactivity (an immunomarker for activated microglia), following nodose ganglionectomy. Male Wistar Kyoto rats underwent unilateral nodose ganglionectomy or sham operation and were treated with saline or minocycline (50 mg/kg i.p.) 12 h before surgery and twice daily after surgery (each 50mg/kg i.p.) for 3 days. Subsequent to nodose ganglionectomy, [125I] CGP42112 binding (insensitive to PD123319 or Ang II) was increased approximately two-fold in the ipsilateral nucleus of the solitary tract and was also induced in the ipsilateral dorsal motor nucleus of the vagus nerve and ambiguus nucleus of saline-treated rats. Treatment with minocycline reduced this non-angiotensin II [125I] CGP42112 binding (40-50% reduction) in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and ambiguus nucleus. Analogous experiments using [3H] PK11195 also revealed up-regulated binding in the ipsilateral nucleus of the solitary tract ( approximately 205%), dorsal motor nucleus of the vagus nerve (approximately 80%) and ambiguus nucleus (approximately 210%) of saline-treated rats following nodose ganglionectomy, which was reduced by 40-100% with minocycline treatment. Immunoreactivity to OX-42 confirmed an increase in microglia activation and accumulation of macrophages in these brain stem nuclei following nodose ganglionectomy, which was also attenuated following treatment with minocycline. These data demonstrate that non-Ang II [125I] CGP42112 binding following nodose ganglionectomy is attenuated by minocycline treatment. This minocycline-induced effect was associated with reduced activation of microglia and an apparent reduction in the number of macrophages in the abovementioned nuclei. This evidence suggests that a non-Ang II [125I] CGP42112 binding site is located on, or associated with, activated microglia and macrophages, providing a useful tool with which to quantitate the neuroprotective effects of centrally acting anti-inflammatory compounds.