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COVID-19 vaccinations are recommended for children with cancer but data on their vaccination response is scarce. This study assesses the antibody and T-cell response following a 2- or 3-dose vaccination with BNT162b2 mRNA COVID-19 vaccine in children (5–17 years) with cancer. For the antibody response, participants with a serum concentration of anti-SARS-CoV-2 spike 1 antibodies of >300 binding antibody units per milliliter were classified as good responders. For the T-cell response, categorization was based on spike S1 specific interferon-gamma release with good responders having >200 milli-international units per milliliter. The patients were categorized as being treated with chemo/immunotherapy for less than 6 weeks (Tx < 6 weeks) or more than 6 weeks (Tx > 6 weeks) before the first immunization event. In 46 patients given a 2-dose vaccination series, the percentage of good antibody and good T-cell responders was 39.3% and 73.7% in patients with Tx < 6 weeks and 94.4% and 100% in patients with Tx > 6 weeks, respectively. An additional 3rd vaccination in 16 patients with Tx < 6 weeks, increased the percentage of good antibody responders to 70% with no change in T-cell response. A 3-dose vaccination series effectively boosted antibody levels and is of value for patients undergoing active cancer treatment.

In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF1 receptor (CRF1R) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gβγ reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gβγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/βγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-Δ386 mutant, which has impaired agonist-dependent β-arrestin-2 recruitment; however, this situation may have resulted from the low β-arrestin expression in the COS-7 cells. When β-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/βγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.

Corticotropin-releasing factor (CRF, nomenclature as agreed by the NC-IUPHAR subcommittee on Corticotropin-releasing Factor Receptors [30]) receptors are activated by the endogenous peptides corticotrophin-releasing hormone, a 41 amino-acid peptide, urocortin 1, 40 amino-acids, urocortin 2, 38 amino-acids and urocortin 3, 38 amino-acids. CRF1 and CRF2 receptors are activated non-selectively by CRH and UCN. CRF2 receptors are selectively activated by UCN2 and UCN3. Binding to CRF receptors can be conducted using radioligands [125I]Tyr0-CRF or [125I]Tyr0-sauvagine with Kd values of 0.1-0.4 nM. CRF1 and CRF2 receptors are non-selectively antagonized by α-helical CRF, D-Phe-CRF-(12-41) and astressin. CRF1 receptors are selectively antagonized by small molecules NBI27914, R121919, antalarmin, CP 154,526, CP 376,395. CRF2 receptors are selectively antagonized by antisauvagine and astressin 2B.

SAR studies were performed on a series of 2-arylamido-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide derivatives as cannabinoid receptor agonists. Starting from a HTS hit both potency and selectivity could be improved. Modifications to the thiophene fusion and C-3 amides were studied. A representative compound 3t produced analgesia when dosed orally in inflammatory pain models of writhing and carrageenan-induced allodynia.

Within the German Plant Phenotyping Network (DPPN), we developed a novel PET scanner based on Philips Digital Photon Counters (DPCs, or dSiPMs = digital Silicon Photomultipliers). The scanner is dedicated for plant research and provides functional information on carbon transport within the plant. To this end the detector ring is oriented horizontally. It provides a Field-of-View of 18 cm dia. and 20 cm in height. The read-out electronics cluster hits from different photodetector pixels when they originate from the same scintillation event. These single events are written via USB 3.0 with up to 300 MB/s to the computer system. Crystal identification, energy discrimination and coincidence detection is realized in software. The spatial resolution in the center Field-of-View (CFOV) could be estimated to approx. 1.6 mm from measurements of a dedicated hot rod phantom. Preliminary sensitivity measurements result in a peak sensitivity of 4.04% (ΔE = 250-750 keV) in the CFOV and a Coincidence Resolving Time of 298 ps could be achieved.

The reasons for differences in vulnerability or resilience to the development of posttraumatic stress disorder (PTSD) are unclear. Here we review key genetic diatheses and molecular targets especially signaling pathways that mediate responses to trauma and severe stress and their potential contribution to the etiology of PTSD. Sensitization of glucocorticoid receptor (GR) signaling and dysregulation of GR modulators FKBP5, STAT5B, Bcl-2, and Bax have been implicated in PTSD pathophysiology. Furthermore, Akt, NFκB, MKP-1, and p11, which are G protein-coupled receptor (GPCR) pathway molecules, can promote or prevent sustained high anxiety- and depressive-like behavior following severe stress. Agonist-induced activation of the corticotropin releasing factor CRF(1) receptor is crucial for survival in the context of serious danger or trauma, but persistent CRF(1) receptor hypersignaling when a threatening or traumatic situation is no longer present is maladaptive. CRF(1) receptor single nucleotide polymorphisms (SNPs) can confer susceptibility or resilience to childhood trauma while a SNP for the PAC1 receptor, another class B1 GPCR, has been linked genetically to PTSD. GRK3 phosphorylation of the CRF(1) receptor protein and subsequent binding of βarrestin2 rapidly terminate Gs-coupled CRF(1) receptor signaling by homologous desensitization. A deficient GRK-βarrestin2 mechanism would result in excessive CRF(1) receptor signaling thereby contributing to PTSD and co-morbid posttraumatic depression. Clinical trials are needed to assess if small molecule CRF(1) receptor antagonists are effective prophylactic agents when administered immediately after trauma. βarrestin2-biased agonists for CRF receptors and possibly other GPCRs implicated in PTSD, however, may prove to be novel pharmacotherapy with greater selectivity and therapeutic efficacy. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Compiling pharmacological evidence implicates metabotropic glutamate mGlu 2 receptors in the regulation of emotional states and suggests positive modulators as a novel therapeutic approach of Anxiety/Depression and Schizophrenia. Here, we investigated subcutaneous effects of the metabotropic glutamate mGlu 2/3 agonist (LY354740) on sleep–wake architecture in rat. To confirm the specific effects on rapid eye movement (REM) sleep were mediated via metabotropic glutamate mGlu 2 receptors, we characterized the sleep–wake cycles in metabotropic glutamate mGlu 2 receptor deficient mice (mGlu 2 R −/− ) and their arousal response to LY354740. We furthermore examined effects on sleep behavior in rats of the positive allosteric modulator, biphenyl-indanone A (BINA) alone and in combination with LY354740 at sub-effective doses. LY354740 (1, 3 and 10 mg/kg) dose-dependently suppressed REM sleep and prolonged its onset latency. Metabotropic glutamate mGlu 2 R −/− and their wild type (WT) littermates exhibited similar spontaneous sleep–wake phenotype, while LY354740 (10 mg/kg) significantly affected REM sleep variables in WT but not in the mutant. In rats, BINA (1, 3, 10, 20, 40 mg/kg) dose-dependently suppressed REM sleep, lengthened its onset latency and slightly enhanced passive waking. Additionally, combined treatment elicited a synergistic action on REM sleep variables. Our findings show common changes of REM sleep variables following modulation of metabotropic glutamate mGlu 2 receptor and support an active role of this receptor in the regulation of REM sleep. The synergistic action of BINA on LY354740's effects on sleep pattern implies that positive modulators would tune the endogenous glutamate tone suggesting potential benefit in the treatment of psychiatric disorders, in which REM sleep overdrive is manifested.

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.

Melanin-concentrating hormone (MCH) is a hypothalamic peptide that centrally regulates food intake, energy balance and emotion. Interestingly, MCH and melanin-concentrating hormone MCH(1) receptors are distributed in brain areas known to regulate vigilance states. Effects of subcutaneous administration of two selective melanin-concentrating hormone MCH(1) receptor antagonists, labeled A and B were examined over a broad dose range (1, 3, 10, 20, 40 mg/kg) on rat sleep-wake architecture. Both compounds have a nanomolar antagonist activity at recombinant human melanin-concentrating hormone MCH(1) receptor (IC(50)=44.1+/-6.1 nM and 26.6+/-5.4 nM, respectively) and potently inhibited the MCH-induced mobilization of [Ca(2+)] (IC(50) 29.1+/-8.1 nM and 10.5+/-4.1 nM, respectively). The selectivity of both compounds was further confirmed on a panel of receptors, transporters and channels. In vivo, both compounds dose-dependently decreased deep sleep primarily by decreasing the mean duration of episodes during the first 4 h post-administration. In parallel, REM sleep and intermediate stage sleep were decreased while active and passive waking increased. Deep sleep and REM sleep onset latencies were significantly prolonged at higher doses. No homeostatic rebound of deep sleep was observed, while a tendency for recovery of REM sleep was found during subsequent dark phase. Together, the results support a role of the melanin-concentrating hormone MCH(1) receptor in the regulation of deep slow-wave sleep-REM sleep cycle. Therapeutic application of melanin-concentrating hormone MCH(1) receptor-inhibiting agents should take into account the significant decreases in deep sleep without recovery as these may interfere with sleep dependent memory consolidation.

A rapid, simple, and efficient method for the preparation of highly dispersed supported Au catalysts has been developed. In the preparation, NaBH 4 is used to reduce the Au precursor, lysine is employed to cap the formed Au colloids, and a short sonication time is applied to facilitate dispersion and deposition of the Au colloids onto the catalyst support, which has been mixed with the precursor beforehand. The end-point pH value of the solution and the isoelectric points (IEPs) of the catalyst supports have an influence on the size of the Au particles and their deposition. The optimum value for the end-point pH is 7.5-10.0, and the IEP should be 5-10. The amino acid capping agent is easily removed at the catalyst activation stage at 200 °C, and the Au particles are thermally stable against sintering, even at 500 °C for 3 h. It is also proven that the method is applicable to the preparation of supported Pt catalysts. The catalytic activity of the prepared Au catalysts for CO oxidation in the absence/presence of H 2 is comparable to that of a Au catalyst prepared by the co-precipitation (CP) method, and to that of the standard catalyst from the World Gold Council (WGC). X-ray photoelectron spectroscopy (XPS) results show that only metallic Au exists in the catalysts before and after activation, and also after the catalysis reaction.

Objectives: The objective of this study was to examine Klebsiella oxytoca clonal and phylogenetic diversity, based on an international collection of carriage isolates non-susceptible to expanded-spectrum cephalosporins (ESCs). Methods: The study material comprised 68 rectal carriage K. oxytoca isolates non-susceptible to ESCs recovered in 2008-11 from patients in 14 hospitals across Europe and Israel. ESC resistance was tested phenotypically; genes encoding ESBLs, AmpC cephalosporinases and carbapenemases were amplified and sequenced. The isolates were typed by PFGE and MLST, followed by sequencing of blaOXY genes. Results: MLSTand PFGE distinguished 34 STs and 47 pulsotypes among the isolates, respectively. Six STswere split into several pulsotypes each. Five STs were more prevalent (n=2-9) and occurred in several countries each, including ST2, ST9 and ST141, which belong to a growing international clonal complex (CC), CC2. Four phylogenetic lineages were distinguished, each with another type of chromosomal OXY-type β-lactamase. Three of these, with OXY-1/-5, OXY-2 types and OXY-4, corresponded to previously described phylogroups KoI, KoII and KoIV, respectively. A single isolate from Israel represented a distinct lineage with a newly defined OXY-7 type. The phylogroups showed interesting differences in mechanisms of ESC resistance; KoI strains rarely overexpressed the OXY enzymes but commonly produced ESBLs, whereas KoII strains often were OXY hyperproducers and carried ESBLs much less frequently. AmpCs (DHA-1) and carbapenemases (VIM-1) occurred sporadically. Conclusions: The study confirmed the high genetic diversity of the collection of K. oxytoca ESC-non-susceptible isolates, composed of phylogroups with distinct types of OXY-type β-lactamases, and revealed some STs of broad geographical distribution.

Several corticotropin-releasing factor (CRF) family genes have been identified in vertebrates. Mammals have four paralogous genes that encode CRF or the urocortins 1, 2, and 3. In teleost fishes, a CRF, urotensin I (a fish ortholog of mammalian urocortin 1) and urocortin 3 have been identified, suggesting that at least three of the four mammalian lineages arose in a common ancestor of modern bony fishes and tetrapods. Here we report the isolation of genes orthologous to mammalian urocortin 1 and urocortin 3 from the South African clawed frog, Xenopus laevis. We characterize the pharmacology of the frog peptides and show that X. laevis urocortin 1 binds to and activates the frog CRF1 and CRF2 receptors at picomolar concentrations. Similar to mammals, frog urocortin 3 is selective for the CRF2 receptor. Only frog urocortin 1 binds to the CRF-binding protein, although with significantly lower affinity than frog CRF. Both urocortin genes are expressed in brain, pituitary, heart, and kidney of juvenile frogs; urocortin 1 is also expressed in skin. We also identified novel urocortin sequences in the genomes of pufferfish, zebrafish, chicken, and dog. Phylogenetic analysis supports the view that four paralogous lineages of CRF-like peptides arose before the divergence of the actinopterygian and sarcopterygian fishes. Our findings show that the functional relationships among CRF ligands and binding proteins, and their anorexigenic actions mediated by the CRF2 receptor, arose early in vertebrate evolution.

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.

Human SMS-KAN neuroblastoma cells endogenously express the neuropeptide Y (NPY) type 2 (Y 2 ) receptor. Although ligand binding and GTPγS binding studies supported high functional Y 2 receptor expression, only weak coupling to the natural second messenger cyclic AMP was observed. The main reason was the low responsiveness of SMS-KAN cells to forskolin, a direct activator of adenylyl cyclases. In order to obtain a cell-based functional assay for the Y 2 receptor in SMS-KAN cells, the transient calcium (Ca 2+ ) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gq i9 . This manipulation resulted in robust mobilization of Ca 2+ after challenge with various NPY-related agonists in a 384-well format. The sensitivity of the FLIPR readout was in the low nanomolar range for NPY agonists and comparable to that of the recombinant Y 2 receptor. The selective Y 2 antagonist BIIE0246 competitively inhibited NPY-mediated Ca 2+ transients in SMS-KAN/Gq i9 cells with a p A 2 value of 7.39 ± 0.1. This is the first evidence that an endogenously expressed G protein-coupled receptor couples to an overexpressed chimeric G protein, thereby functionally responding in the FLIPR readout.

A newly developed γ-ray computed tomography scanner was implemented to study liquid flow distribution in a 30.48-cm-diameter column packed with corrugated structure using countercurrent air–water flow. Validation experiments confirm that spatial resolution of ∼2.5 mm can be achieved by the new CT unit. Within the gas (0–10 cm/s superficial velocity) and liquid (0.6–2.5 cm/s superficial velocity) flow ranges investigated, a good liquid distribution was observed at all conditions, as manifested by uniformity factor in excess of 70%. The liquid saturation increases with increasing superficial liquid velocity as well as down the column height. Within the conditions studied, the effect of gas velocity was, in general, found to be minimal.

Neuropeptide Y (NPY) receptors type 1 (Y1), type 2 Y2) and type 5 (Y5) were tested for their kinetic properties to bind radiolabeled NPY or PYY. Rapid association and dissociation was observed with recombinant (HEK293 cells) and endogenous (SK-N-MC cells) human Y1 and recombinant mouse Y5 receptors. Recombinant (HEK293) and endogenous (SMS-KAN) human Y2 receptors bound both radiolabels comparable to the Y1 receptors, but only minimal (∼20%) dissociation of both radiolabels was observed after long incubation time (>8 h). Furthermore, neither peptide nor small molecule Y2 ligands efficiently competed for binding to Y2 receptors once association binding had been initiated. The Y2-selective antagonist BIIE0246 behaved as an insurmountable antagonist in functional assays when pre-incubated for 30 min before agonist addition, but was a competitive antagonist when co-applied with the agonist. These data show that Y2 receptors in contrast to Y1 and Y5 receptors bind their ligands in an irreversible manner.

In order to promote the introduction of emerging technologies in the process industry, it has been established the two types of supporting RD and (2) multi‐functional reactors by integrating catalysis, heat transfer and/or separation. Catalyst Synthesis: In this paper, several methods of catalyst tailoring will be described that can minimize mass transfer limitations at industrially relevant conversion levels. Three (3) specific examples have been selected to demonstrate what can be achieved: (1) micro‐engineered catalyst that enables enhanced inter‐phase transfer; (2) new mesoporous catalysts with ultra large pores to accommodate slowly diffusing reactants; and (3) customized zeolites of extremely small particles to achieve high effectiveness factors while retaining the virtues of shape selectivity. Multi‐functional Reactors: Applying process intensificatio...

The human corticotropin-releasing factor (hCRF) receptors CRF 1 and CRF 2(a) couple to the G s protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to G s in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF 1 and hCRF 2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose–response curves. Complete inhibition of calcium signaling of both hCRF 1 and hCRF 2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP 3 ) accumulation in hCRF 1 -and hCRF 2(a) -transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.

Catalysis plays a critical role in virtually every industry. Often it is the key to making an entirely new technology or breathing new life into an otherwise, mature technology. In addition to the continued needs for productivity improvements, environmental drives and heightened industrial safety add a new aspect to the importance of catalytic innovation. ABB Lummus Global, along with its many industrial partners, has a well-established, track record of fostering innovation in its many technologies. These innovations take form in many ways: (a) catalyst design, (b) advanced materials, (c) new process concepts, (d) engineering advances, and (e) novel uses of high throughput screening. Moving projects through the “technology pipeline” used to be done by brute force–using an army of scientists and engineers. Today that luxury no longer exists, and industry must embrace creative approaches to leverage their resources and accelerate the development and commercialization of new technology. This paper presents specific examples of innovation at Lummus. These examples will, once again, reinforce the point that catalytic innovation is clearly an important vehicle to continued advancement of society.

The neuropeptides orexin A and B (also known as hypocretins) play an important role in many physiological and behavioral activities. Orexins are ligands of two closely related G-protein-coupled receptors, that are the named orexin 1 and orexin 2 receptors. To clearly identify the minimal ligand sequences required for receptor activation, we synthesized and analyzed different centrally, C- and N-terminally truncated analogues of orexins A and B. Furthermore, we used the shortest active analogue to screen for important amino acid residues by l-alanine and l-proline replacement scans. For orexin A, only full-length peptides were able to show the same activity as orexin A, but interestingly, reduced orexin A and natural orexin A, which contains the two disulfide bonds, had the same activity. The shortest highly active orexin B analogue was orexin B 6-28. In addition, we identified orexin A 2-33 as the first analogue with orexin 1 receptor preference and orexin B 10-28, [A27]orexin B 6-28, and [P11]orexin B 6-28 as being highly potent orexin 2 receptor selective (1000-fold) peptides.

Protein kinase C (PKC)-mediated desensitization of the corticotropin releasing factor type 1 (CRF1) receptor was investigated in human retinoblastoma Y79 and transfected COS-7 cells. Because stimulation of Y79 cells with CRF resulted in large ( approximately 30-fold) increases in intracellular cAMP accumulation without changing inositol phosphate levels, the CRF1 receptor expressed in retinoblastoma cells couples to Gs, but not to Gq, and predominantly signals via the protein kinase A cascade. Direct activation of PKC by treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) desensitized CRF1 receptors in Y79 cells, reducing the maximum for CRF- (but not forskolin)-stimulated cAMP accumulation by 56.3 +/- 1.2% and 40.4 +/- 2.1%, respectively (p < 0.001). Pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (BIM) markedly inhibited PMA's desensitizing action on CRF-stimulated cAMP accumulation, but did not affect homologous CRF1 receptor desensitization. Retinoblastoma cells were found to express PKCalpha, betaI, betaII, delta, lambda, and RACK1. When alpha and beta isoforms of PKC were down-regulated 80 to 90% by a 48-h PMA exposure, PMA-induced CRF1 receptor desensitization was abolished. In transfected COS-7 cells the magnitude of CRF1 receptor phosphorylation after a 5-min exposure to PMA was 2.32 +/- 0.21-fold greater compared with the basal level. Pretreating COS-7 cells with BIM abolished PMA-induced CRF1 receptor phosphorylation. These studies demonstrate that protein kinase C (possibly alpha and beta isoforms) has an important role in the phosphorylation and heterologous desensitization of the CRF1 receptor.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

A kinetic study into the styrene hydrogenation over a palladium on alumina catalyst has been made. Styrene was used as a model component for pyrolysis gasoline. A kinetic rate expression has been derived and the inhibiting effect of sulfur components has been included. Using this kinetics and mass-transfer models compiled from literature, the performance of two types of reactors for the styrene (pyrolysis gasoline) hydrogenation has been evaluated. A structured reactor such as a monolith has large advantages over a conventional trickle-bed reactor. For the monolithic reactor a more than 3 times higher volumetric productivity is obtained with much less catalyst. The modeling results indicate that deactivation by gum formation should be significantly less due to much better hydrogen mass transfer in the reactor.

Receptors for corticotropin-releasing factor (CRF) are members of a family of G protein-coupled receptors ("Family B") that respond to a variety of structurally dissimilar releasing factors, neuropeptides, and hormones (including secretin, growth hormone-releasing factor, calcitonin, parathyroid hormone, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal polypeptide) and signal through the cyclic AMP and/or calcium pathways. To date, three genes encoding additional CRF-like peptides (urocortins) have been identified in mammals. The urocortins and CRF bind with differential ligand selectivity at the two mammalian CRF receptors. This report was prepared by the International Union of Pharmacology Subcommittee on CRF Receptors, to summarize the current state of CRF receptor biology and to propose changes in the classification and nomenclature of CRF ligands and receptors.

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF 2 ) receptor. A structure–activity relationship study was performed to pinpoint residues conferring aSVG’s selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala 22 Arg 23 Ala 24 (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for α-helicity properties, binding affinities and antagonistic potencies at the rat CRF 1 and mouse CRF 2B receptors. While N-terminal prolongation and replacement of d -Phe 11 by Tyr 11 increased the affinity for the CRF 2 receptor, the introduction of the ARA motif resulted in a loss of CRF 2 receptor selectivity. These data show that aSVG 10–40 analogues are more potent CRF 2 receptor antagonists than aSVG 11–40 peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF 1 receptor.

Novel analogues of antisauvagine-30 (aSvg-30), a selective antagonist for CRF2 receptors, have been synthesized and characterized in vitro and in vivo. The analogues were tested for their ability to compete for [125I-Tyr0]Svg binding and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF1 (hCRF1), hCRF2α and hCRF2β receptor. One analogue [D-Phe11, His12, Nle17]Svg(11-40), named K41498, showed high affinity binding to hCRF2α (Ki=0.66±0.03 nM) and hCRF2β (Ki=0.62±0.01 nM) but not the hCRF1 receptor (ki=425+50 nM) and decreased Svg-stimulated cAMP accumulation in hCRF2 expressing cells. In conscious Wistar-Kyoto rats, K41498 (1.84 μg, i.v.) antagonized the hypotensive response to systemic urocortin (1.4 μg, i.v.), but did not block the pressor response to centrally administered urocortin (2.35 μg, i.c.v.). K41498 was subsequently radio-iodinated, and in autoradiographic studies, specific (sensitive to rat urocortin, astressin and aSvg30, but insensitive to antalarmin) binding of 125I-K41498 (100 pM) was detected in the heart and in selected brain regions including the nucleus tractus solitarius (NTS), spinal trigeminal nucleus, lateral septum and around the anterior and middle cerebral arteries. Following unilateral nodose ganglionectomy, binding of 125I-K41498 was reduced by 65% in the ipsilateral NTS, indicative of presynaptic CRF2 receptors on vagal afferent terminals. These data demonstrate that K41498 is a useful tool to study native CRF2 receptors in the brain and periphery. British Journal of Pharmacology (2002) 136, 896–904. doi:10.1038/sj.bjp.0704783

Catalytic cracking reactions of n-hexane over molybdenum oxide (MoO2) grown on a polycrystalline molybdenum foil (∼1 cm2) were studied in the temperature range 830–913 K at a low conversion (

The ligand selectivity of human (hCRF(2A)) and Xenopus laevis (xCRF(2)) forms of the corticotropin-releasing factor type 2 (CRF(2)) receptor differs. The purpose of this study was to identify amino acids in these two CRF(2) receptors conferring these differences. An amino acid triplet in the third extracellular domain (Asp(262)Leu(263)Val(264) in hCRF(2A) or Lys(264)Tyr(265)Ile(266) in xCRF(2)) was found to diverge between both receptors. When binding and signaling characteristics of receptor mutants hR2KYI and xR2DLV were assessed, the tri-amino acid motif replacement produced receptors with binding properties resembling the xCRF(2) receptor. The converse mutation created a mutant receptor with a binding pharmacology identical to the profile of the hCRF(2A) receptor. This effect was most notable for xR2DLV, which possessed a binding affinity for astressin approximately 15-fold greater for astressin than sauvagine. In contrast, the binding profiles of the hCRF(2A) receptor and hR2KYI did not differ. These data indicate that another domain of the xCRF(2) receptor mediated low-affinity binding of astressin. Two amino acids in the first extracellular domain differ in xCRF(2) (Asp(69)Ser(70)) and hCRF(2A) (Glu(66)Tyr(67)) receptors. The hCRF(2A) receptor mutant (hR2DS-KYI) bound astressin with a low affinity indistinguishable from the xCRF(2) receptor. Therefore, these data demonstrate that ligand selectivity differences between amphibian and human forms of the CRF(2A) receptor are governed by these two motifs of the extracellular domains of the xCRF(2) receptor.

Three full-length cDNAs encoding functional splice variants of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) were isolated from Y-79 retinoblastoma cells and human cerebellum. Although the third intracellular loops of the three splice variants were identical, their N-terminal extracellular domains differed. The first full-length PAC1 variant, PAC1normal (PAC1n), encoded the entire N-terminus, whereas the second variant named PAC1short (PAC1s) was deleted by 21 amino acids (residues 89-109). Finally, the third variant, named PAC1very short (PAC1vs), was deleted by 57 amino acids (residues 53-109). Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, it was established that all three variants were expressed in neuronal tissues. Binding- and cAMP studies using human embryonic kidney 293 (HEK293) cells stably transfected with PAC1n, PAC1s and PAC1vs showed significant differences in the affinities and selectivities towards PACAP38, PACAP27 and VIP. PAC1n bound PACAP38 and PACAP27 with affinities in the low nanomolar range whereas VIP was bound with up to 400-fold lower affinity. PAC1vs preferentially bound PACAP38 (Ki=121 nM) and PACAP27 (Ki=129 nM) over VIP (Ki>1000 nM) but with 100-fold lower affinity than PAC1n. Surprisingly, PAC1s unselectively bound all three ligands with high affinity. These data indicate that residues 53-88 within the N-terminal domain of the PAC1 are important for high affinity ligand binding, whereas residues 89-109 determine the receptor's ligand selectivity.

ABB has advanced catalysis with micro-engineered catalyst (MEC) systems by providing a uniquely small particle size on a formable catalyst support through the integration of catalysis and reaction engineering. A mechanically strong catalytic web of micro-fibers has been engineered and shaped utilizing both computational fluid dynamics (CFD) and cold flow experiments to optimize flow characteristics. This article discusses techniques used for the development of novel catalytic structured packings for catalytic distillation applications. CFD models (verified through experiments performed on small-sized structures) were shown to be of great utility in screening new structure ideas. Results will illustrate achievement of both high gas–liquid contacting and bulk mixing at low pressure drop with the potential to provide enhanced catalyst utilization by taking advantage of the intrinsic MEC properties, particularly its high porosity and exposed geometric fiber and catalyst surface area. This was shown by the successful testing of one of these catalyzed structures in the selective hydrogenation of C4 acetylenes.

Dysfunctioning of corticotropin-releasing hormone (CRH) and its receptors (CRH 1 and CRH 2 ) has been linked to the development of stress-related disorders, such as mood and eating disorders. The molecular characterization of CRH 1 and CRH 2 receptors and their splice variants has generated detailed information on their pharmacology, tissue distribution and physiology. While mammalian CRH 1 receptors nonselectively bind CRH analogs, the ligand specificity of CRH 2 is narrower. CRH 1 receptors are predominantly expressed in the brain and pituitary, whereas CRH 2 receptor expression is limited to particular brain areas and to some peripheral organs. Molecular approaches to block CRH 1 receptor expression in the brain argue in favor of its involvement in the regulation of some aspects of the stress response. The CRH 2α receptor may be more important for motivational types of behavior essential for survival, such as feeding and defense. 1

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI5–22or H-89 failed to attenuate homologous desensitization of CRF1receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1receptors by ∼35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by ∼50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1receptors by ∼55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an ∼50% reduction in GRK3 protein expression and an ∼65% reduction in homologous desensitization of CRF1receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1receptors in Y-79 cells, a brain-derived cell line.

Corticotropin-releasing factor (CRF) receptors type 1 (CRF(1)) and type 2 (CRF(2)) differ from each other in their pharmacological properties. The human and ovine CRF versions bind to CRF(1) receptors with significantly higher affinity than to CRF(2) receptors. Recently antisauvagine-30, an N-terminally truncated version of the CRF analog sauvagine, was characterized as a specific antagonist to mouse CRF(2B). We have synthesized the radiolabeled version (125)I-antisauvagine-30 and tested it for its affinity at human CRF(1) (hCRF(1)), hCRF(2A), Xenopus CRF(1) (xCRF(1)) and xCRF(2) receptors. In control binding studies (125)I-labeled hCRF, sauvagine and astressin were also bound to these receptors. (125)I-antisauvagine-30 exclusively bound to hCRF(2A) and xCRF(2) but not to hCRF(1) and xCRF(1) receptors. (125)I-antisauvagine-30 binding to hCRF(2A) and xCRF(2) receptors was saturable and of high affinity (hCRF(2A): K(d)=125 pM; xCRF(2): K(d)=1.1 nM). In displacement binding experiments using (125)I-antisauvagine-30 as radioligand several CRF analogs bound to hCRF(2A) and xCRF(2) receptors with similar rank orders as reported with other CRF radioligands. Finally, preliminary studies using (125)I-antisauvagine-30 binding to membrane homogenates prepared from different rat brain structures showed that the peptide bound specifically to brain areas expressing CRF(2) receptors. These data demonstrate that (125)I-antisauvagine-30 is the first high-affinity ligand to specifically label CRF(2) receptors.

The development of 8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3, 8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (micro, kappa, delta) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPgamma(35)S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.

The abundance of a histidine residue at position 185 (His(185)) of the human corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2alpha)) was investigated. His(185) has only been reported in hCRF(2); CRF(2) proteins from other species and all CRF(1) receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) cDNAs from a variety of neuronal and peripheral tissues revealed the existence of receptor molecules encoding Arg(185) only. Sequence analysis of the hCRF(2) gene verified the existence of Arg(185) also on genomic level. Full-length cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185)) variants of hCRF(2alpha) were stably expressed in HEK293 cells and tested for ligand binding properties. In displacement studies R2H(185) and R2R(185) displayed a similar substrate specificity, human and rat urocortin, and the peptide antagonists astressin and alpha-helical CRF((9-41)) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. Significant differences were observed for sauvagine and urotensin I, which bound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2R(185). These data indicate that hCRF(2), like all vertebrate CRF(1) and CRF(2) proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role for the discrimination of some CRF peptide ligands.

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF ( K i =3.6 nM), ovine CRF ( K i =4.6 nM), rat urocortin ( K i =2.2 nM), sauvagine ( K i =2.4 nM) and astressin ( K i =4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF 1 ) receptors and B max values of ∼30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose–response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF 1 transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF 2 could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF 1 receptors.

ResumenEn el presente estudio se valoraron dos hipótesis generales sobre los orígenes de la automu-tilación en los pacientes psiquiátricos. La primera afirma que la automutilación tiene su origen en el abuso infantil y las experiencias de abandono y está relacionada con disociación en momentos posteriores de la vida. La segunda ve la automutilación como consecuencia de problemas del control de impulsos. Para examinar estas dos hipótesis, se recogieron datos acerca de experiencias infantiles traumáticas y síntomas disociativos (hipótesis 1), así como datos acerca de la agresividad, la conducta obsesivo-compulsiva y la búsqueda de sensaciones (hipótesis 2) en una muestra de 54 pacientes psiquiátricos hospitalizados. Veinticuatro de 54 pacientes (44%) comunicaron haberse mutilado. La edad media de comienzo de este comportamiento era 23 años. Las medidas de autoinforme de los pacientes que se mutilaron estaban más de acuerdo con la primera hipótesis que con la segunda. Es decir, los pacientes que se mutilaron comunicaban más experiencias infantiles traumáticas y síntomas disociativos que los pacientes de control. Los dos grupos no diferían por lo que se refiere a la agresividad, la conducta obsesivo-compulsiva y la búsqueda de sensaciones. De acuerdo con estudios anteriores, los presentes resultados indican que el comportamiento de automutilación se relaciona con una historia de abuso y abandono.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.

From brain, heart and muscle tissue of the tree shrew (Tupaia belangeri), a higher order mammal, cDNA clones were isolated that encoded two functional splice variants of the corticotropin-releasing factor (CRF) type 2 receptor (CRF-R2). The first, full-length splice variant, amplified from brain and heart tissue, encoded a CRF receptor protein that is 410 amino acids in length and approximately 96% homologous to human CRF-R2alpha. The second, full-length splice variant, derived from skeletal muscle tissue, encoded a 437-amino acid CRF receptor protein that is approximately 92% homologous to human CRF-R2beta. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) amplifications and RNase protection analyses, showed that tree shrew CRF-R2alpha (tCRF-R2alpha) and tree shrew CRF-R2beta (tCRF-R2beta) were coexpressed in brain tissue but not in heart and skeletal muscle tissue. Finally, human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R2alpha and tCRF-R2beta were used to demonstrate that the CRF analogs urocortin and sauvagine bind with significantly greater affinity (21- to 140-fold) to these two CRF-R2 splice variants than do human/rat and ovine CRF analogs. In keeping with these results of our CRF binding studies, EC50 values were substantially lower for urocortin-and sauvagine-stimulated than for h/rCRF-and oCRF-stimulated cyclic AMP accumulation in HEK293 cells stably transfected with tCRF-R2alpha or tCRF-R2beta cDNAs. The tree shrew therefore constitutes an important animal model in which to investigate the role of CRF receptor subtypes in the stress response.

SummaryIn the present study two broad hypotheses about the origins of self-mutilation in psychiatric patients were evaluated. The first hypothesis states that self-mutilation originates from child abuse and experiences of neglect and is connected to dissociation in later life. The second hypothesis views self-mutilation as the consequence of impulse control problems. To test these two hypotheses, data concerning traumatic childhood experiences and dissociative symptoms (hypothesis 1), as well as data concerning aggressiveness, obsessive-compulsiveness and sensation seeking (hypothesis 2) were collected in a sample of 54 psychiatric inpatients. Twenty-four out of 54 patients (44%) reported having engaged in self-mutilation. Mean age of onset of this behaviour was 23 years. Self-report measures of self-mutilators were more in line with the first than with the second hypothesis. That is, patients who engaged in self-mutilation reported more traumatic childhood experiences and dissociative symptoms than did control patients. The two groups did not differ in terms of aggressiveness, obsessive-compulsiveness, and sensation seeking. In line with earlier studies, the current results indicate that self-mutilating behaviour is linked to a history of abuse and neglect.

The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg76 or Asn81 but not Gly83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg 76 , Asn 81 , Gly 83 , Leu 88 , and Ala 89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln 76 , Gly 81 , Val 83 , His 88 , and Leu 89 ) resulted in a receptor that had ∼30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (∼2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg 76 , Asn 81 , and Gly 83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln 76 , Gly 81 , and Val 83 ) resulted in a receptor with ∼11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70–89 of CRF-R1 are important for the ligand binding site.

The alkylation of benzene with light hydrocarbons over an acidic catalyst is an important industrial process. Metal halides and solid phosphoric acid have been the most commonly used acid catalysts for this purpose, while zeolite catalysts are excellent, environmentally clean alternatives. Although liquid-phase alkylation with certain synthetic zeolites has been established commercially (such as in catalytic distillation), little has been published on the fundamental parameters governing the reaction kinetics. This paper examines the effect of external (liquid−solid) and internal (pore diffusion) mass transfer on the main reaction of benzene alkylation with ethylene and propylene using various zeolite catalysts. The results and its implications on the effective catalyst utilization will be presented. This constitutes one of the series of basic studies in an effort to optimize aromatic alkylation reactor systems such as catalytic distillation.

The significance of different domains of corticotropin-releasing factor receptor, type 1, (CRFR1) for ligand binding and cAMP accumulation was investigated with C-terminally truncated forms of rat CRFR1 (rCRFR1) tagged by a sequence of six histidine residues (His-tag) to facilitate protein purification and identification. These different forms of the receptor were N-glycosylated and transported properly to the membranes of transfected mammalian cells as indicated by Western blot analysis and immunocytochemical staining with two polyclonal antibodies developed against the N- and C-terminus of rCRFR1. The N-terminal fragment, rCRFR1(23-121), expressed in Escherichia coli bound oCRF specifically, but with low affinity. Several mutants lacking transmembrane domain (TM) 7 and the C-terminus exhibited similarly low affinities to oCRF after expression in transfected mammalian cells. None of these cells produced significant amounts of cAMP after exposure to oCRF. Only mutants containing the N-terminus, all loops and TMs bound oCRF and produced cAMP with high affinity (Kd = 62 nM) and efficacy (EC50 = 0.8 nM). The additional presence of the C-terminus provided similar characteristics (Kd = 5 nM, EC50 = 0.3 nM) as known for the native receptor. It is suggested on the basis of these data that the last extracellular loop is involved in ligand binding.

The primary goal was to determine agonist-specific regulation of CRF 2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF 2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF 1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF 2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF 2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF 1 receptor signaling. In transfected HEK293 cells, activation of CRF 2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF 2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100 nM) produced strong βarrestin2 translocation and colocalization with membrane CRF 2(a) receptors while CRF (1 μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF 2(a) receptor–arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF 2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF 2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF 2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.