Biomedical subjects

R Lang

Publications and source records attributed to R Lang.

At least 19 recordsLinked to original sources

PubMed2006

Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.

Crystallography, X-Ray↗

PubMed2006

Obesity and disability - a short review.

The prevalence of both obesity and disability is increasing globally and there is now growing evidence to suggest that these two health priorities may be linked. This paper explores the evidence linking obesity to muscular-skeletal conditions, mental health disorders and learning disabilities in both adult and child populations. The impact of obesity on the four most prevalent disabling conditions in the UK (arthritis, mental health disorders, learning disabilities and back ailments) has been examined through novel data analysis of the 2001 Health Survey for England and UK Back Exercise And Manipulation trial data. Together these analyses strongly suggest that whether the cause or result of disability, obesity is undeniably implicated, thus presenting a serious public health priority. Future research efforts are required to strengthen the evidence base examining obesity in back disorders, mental health and learning disabilities, in order to improve current clinical management.

Adult↗

PubMed2006

Hymenoptera venom immunotherapy and field stings.

BACKGROUND: Anaphylactic sting reactions in patients with Hymenoptera venom allergy are prevented by venom immunotherapy (VIT) in most patients. OBJECTIVE: The aim of this study was to investigate re-sting reactions in the field during VIT or during an observation period of up to 13 years after cessation of treatment. Furthermore we sought to identify patients at higher risk of developing a systemic allergic reaction (SAR) and to assess possible correlations with basal serum tryptase concentration. METHODS: The clinical data of 192 patients with a recorded field sting during VIT were evaluated and the patients were questioned regarding possible re-stings after cessation of VIT. Baseline mast-cell tryptase concentrations and specific IgE were analyzed in patients with a reported SAR. RESULTS: Of 192 patients with reported re-stings in the field, 27 developed SARs (14.1%). A SAR occurred in 11.9% of the stings delivered during VIT, whereas 9.7% of the stings resulted in a SAR after VIT. The majority of SARs in response to a field sting during VIT were mild, whereas severe SARs occurred more often after VIT and repeated reexposure. Out of 23 patients with reported SARs, 2 (8.7%) had elevated basal serum tryptase. CONCLUSIONS: VIT lasting for at least 3 years is effective in protecting the vast majority of patients. The individual predictability of the response of patients to a field sting is low. SARs of increased severity mainly occur after therapy and after tolerating consecutive stings.

Adolescent↗

PubMed2005

Polymorphisms in exon 13 and intron 14 of the RET protooncogene: genetic modifiers of medullary thyroid carcinoma?

CONTEXT: Single-nucleotide polymorphisms (SNPs) of the RET protooncogene (RET) could modify disease susceptibility and clinical phenotype in patients with sporadic or familial medullary thyroid carcinoma (FMTC). OBJECTIVE/DESIGN OF THE STUDY: Because frequencies of RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for medullary thyroid carcinoma (MTC), we studied RET SNPs in patients with FMTC (n = 22), patients with sporadic MTC (n = 45), and 71 subjects presenting with moderately elevated hCt concentrations (basal, >10 pg/ml; pentagastrin stimulated, > 50 < 100 pg/ml) in comparison with an age- and gender-matched control group (n = 79) with basal hCt concentrations in the normal range (<5 pg/ml). METHODS: After DNA extraction from citrated whole blood, RET exons 10, 11, 13, 14, 15, and 16 and exon/intron boundaries were analyzed by PCR-based cycle sequencing for RET germ line mutations, exonic (G691S, L769L, S836S, S904S) and intronic (IVS13+158; NCBI rs2472737 = IVS14-24) SNPs. RESULTS: In FMTC patients, the F791Y mutation was found to be associated (P = 0.001) with the L769L SNP. The exonic SNPs (G691S, L769L, S836S, and S904S) were not different among the four groups. The intron 14 SNP (IVS14-24), however, was more frequent in individuals with elevated hCt serum concentrations (P = 0.016) and patients with sporadic MTC (P < 0.001) when compared with the control group. CONCLUSIONS: These data suggest that the exon 13 (L769L) and the intron 14 (IVS14-24) SNPs could act as genetic modifiers in the development of some forms of hereditary and sporadic MTC, respectively.

Adult↗

PubMed2005

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism.

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.

Amino Acid Sequence↗

PubMed2005

Unexpected hospital-acquired bacteraemia in patients at low risk of bloodstream infection: the role of a heparin drip.

Following a cluster of cases of unexpected hospital-acquired bacteraemia suspected to be related to an intravenous (iv) heparin drip, all cases of hospital-acquired primary bloodstream infection (BSI) in patients at low risk of bacteraemia were analysed over a four-year period. Ninety-six bacteraemic patients (6%) from 1618 episodes of hospital-acquired bacteraemia had a peripheral iv line as the only risk factor. These patients were divided into two groups: 60 patients with phlebitis and 36 without local signs of inflammation. Baseline features of the two groups were comparable, but in univariate and multivariate analysis, a significant association was found between iv heparin use, predominance of Gram-negative organisms (especially Klebsiella, Serratia and Enterobacter species), and absence of phlebitis. In spite of clear statistical association, however, the means by which the heparin solution became contaminated with Gram-negative organisms remained unknown. Following implementation of infection control methods concerning heparin handling, no more cases occurred. Unexpected hospital-acquired Gram-negative bacteraemia in patients with peripheral iv lines should prompt investigation of potential infusate-related infection, especially in patients without phlebitis and those receiving iv heparin.

Aged↗

PubMed2004

Lifetime and predissociation yield of 14N2 b 1Piu(v=1).

The lifetime of the b 1Piu(v=1) state in 14N2 has been determined experimentally using a laser-based pump-probe scheme and an exceptionally long lifetime of 2.61 ns was found. Semiempirical close-coupling calculations of the radiative lifetime, which include Rydberg-valence interactions in the singlet manifold, are consistent with this large value, giving a value of 3.61 ns and suggesting a predissociation yield of approximately 28% for this level of the b state.

Journal Article↗

PubMed2004

Crystal structure of the catalytic domain of MMP-16/MT3-MMP: characterization of MT-MMP specific features.

Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3-MMP exhibits a classical MMP-fold with similarity to MT1-MMP. Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1' specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3-MMP involved in the interaction with the proMMP-2 prodomain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2.

Amino Acid Sequence↗

PubMed2004

Marginal adaptation in dentin of a self-adhesive universal resin cement compared with well-tried systems.

OBJECTIVES: This study compares the marginal adaptation of a new self-adhesive universal resin cement with only one application step, to the marginal adaptation of established cements and their corresponding adhesive systems. METHODS: All-ceramic crowns were inserted on human molars using a new self-adhesive universal resin cement without and with one pre-treatment step, a resin cement with a smear-layer removing and a compomer cement with a smear-layer dissolving adhesive system. After simulation of five years oral stress, the marginal adaptation was evaluated by dye penetration and scanning electronic microscope analysis using the replica technique. RESULTS: Scanning electron microscopy: all investigated luting agents showed comparable amounts of 'perfect margin' ranging between 88-98% (median). Dye penetration: the self-adhesive system had significantly lower dye penetration (18-25%, median). SIGNIFICANCE: The results indicate that a self-adhesive universal resin cement without pre-treatment can provide a marginal adaptation at dentin which is comparable to established luting agents.

Coloring Agents↗

PubMed2004

Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during Helicobacter pylori infection.

BACKGROUND AND AIMS: Recent studies linked cytokine gene polymorphisms to H pylori related gastric cancer development. The current study evaluated the role of cytokine gene polymorphisms for mucosal cytokine expression, the gastric inflammatory response, and bacterial colonisation during H pylori infection. PATIENTS AND METHODS: In 207 H pylori infected patients with chronic gastritis, polymorphisms at different loci of the interleukin (IL)-10, IL-1B, IL-1 receptor antagonist (IL-1RN), tumour necrosis factor (TNF)-A, and interferon (IFN)-G genes were genotyped by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and allelic discriminating TaqMan PCR. Mucosal cytokine mRNA copy numbers were determined by real time quantitative PCR. Presence of bacterial virulence factors was investigated by cagA, vacAs1/2, and babA2 PCR. Biopsies were assessed with regard to the degrees of granulocytic/lymphocytic infiltration and the presence of intestinal metaplasia (IM) and atrophic gastritis (AG). RESULTS: Proinflammatory IL-1 polymorphisms (IL-1RN*2(+)/IL-1B-511T/-31C(+)) were associated with increased IL-1beta expression, more severe degrees of inflammation, and an increased prevalence of IM and AG. Carriers of the IL-10-1082G/-819C/-592C alleles (GCC haplotype) had higher mucosal IL-10 mRNA levels than ATA haplotype carriers and were associated with colonisation by more virulent cagA(+), vacAs1(+), and babA2(+) H pylori strains. The TNF-A-307(G/A) and IFN-G+874(A/T) polymorphisms did not influence mucosal cytokine expression or the inflammatory response to H pylori. CONCLUSIONS: Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and the long term development of precancerous lesions in H pylori infection. Host polymorphisms are associated with certain bacterial strain types, suggesting host specific colonisation or adaptation. These findings contribute to the understanding of the complex interplay between host and bacterial factors involved in the development of gastric pathology.

Adult↗

PubMed2004

Superiority of letrozole to tamoxifen in the first-line treatment of advanced breast cancer: evidence from metastatic subgroups and a test of functional ability.

PURPOSE: The letrozole study 025 is a large (n = 907), international, double-blind, randomized, phase III trial in postmenopausal women with advanced breast cancer. This subanalysis compares the efficacies of letrozole and tamoxifen as first-line therapy in postmenopausual women with advanced breast cancer according to site of metastatic lesions and Karnofsky Performance Status (KPS). MATERIALS AND METHODS: Nine hundred seven patients with advanced breast cancer were randomly assigned to once-daily oral letrozole (2.5 mg; Femara; Novartis Pharma AG; Basel, Switzerland) or tamoxifen (20 mg; Tamofen; Leiras OY; Turku, Finland). Time to progression (TTP) was estimated using the Kaplan-Meier product-limit method. Treatments were compared by Cox proportional hazards regression models. RESULTS: Letrozole treatment significantly prolonged TTP in all subsets of patients: those with nonvisceral metastases, those with visceral metastases without liver involvement, and those with liver metastases. The reduction in risk of progression ranged from 25%, for patients with nonvisceral metastases, to 36%, for patients with liver metastases. The distributions of baseline KPS scores for both treatment groups were similar (57% had KPS scores >/=90). Time to worsening of 20 points or more in KPS score was significantly longer with letrozole than with tamoxifen, but modest numbers of patients experienced such deterioration (letrozole, 20%, tamoxifen, 22%, in patients without visceral metastases; 23%-24% in patients with liver metastases; and letrozole, 14%, tamoxifen, 30%, in patients with visceral metastases without liver involvement). CONCLUSION: These data demonstrate the consistent superiority of letrozole over tamoxifen and support the use of letrozole as a new standard of endocrine therapy in postmenopausal women with advanced breast cancer.

Adult↗

PubMed2003

An open randomised trial of second-line endocrine therapy in advanced breast cancer. comparison of the aromatase inhibitors letrozole and anastrozole.

It was previously shown that letrozole (Femara) was significantly more potent than anastrozole (Arimidex) in inhibiting aromatase activity in vitro and in inhibiting total body aromatisation in patients with breast cancer. The objective of this study was to compare letrozole (2.5 mg per day) and anastrozole (1 mg per day) as endocrine therapy in postmenopausal women with advanced breast cancer previously treated with an anti-oestrogen. This randomised, multicentre and multinational open-label phase IIIb/IV study enrolled 713 patients. Treatment was for advanced breast cancer that had progressed either during anti-oestrogen therapy or within 12 months of completing that therapy. Patients had tumours that were either positive for oestrogen and/or progesterone receptors (48%) or of unknown receptor status (52%). The primary efficacy endpoint was time to progression (TTP). Secondary endpoints included objective response, duration of response, rate and duration of overall clinical benefit (responses and long-term stable disease), time to treatment failure, and overall survival, as well as general safety. There was no difference between the treatment arms in TTP; median times were the same for both treatments. Letrozole was significantly superior to anastrozole in the overall response rate (ORR) (19.1% versus 12.3%, P=0.013), including in predefined subgroups (receptor status-unknown, and soft-tissue- and viscera-dominant site of disease). There were no significant differences between the treatment arms in the rate of clinical benefit, median duration of response, duration of clinical benefit, time to treatment failure or overall survival. Both agents were well tolerated and there were no significant differences in safety. These results support previous data documenting the greater aromatase-inhibiting activity of letrozole and indicate that advanced breast cancer is more responsive to letrozole than to anastrozole as second-line endocrine therapy.

Adult↗

PubMed2003

Nutritional management of rheumatoid arthritis: a review of the evidence.

Rheumatoid arthritis (RA) is a debilitating disease and is associated with increased risk of cardiovascular disease and osteoporosis. Poor nutrient status in RA patients has been reported and some drug therapies, such as nonsteroidal anti-inflammatory drugs (NSAIDs), prescribed to alleviate RA symptoms, may increase the requirement for some nutrients and reduce their absorption. This paper reviews the scientific evidence for the role of diet and nutrient supplementation in the management of RA, by alleviating symptoms, decreasing progression of the disease or by reducing the reliance on, or combating the side-effects of, NSAIDs. Supplementation with long-chain n-3 polyunsaturated fatty acids (PUFA) consistently demonstrates an improvement in symptoms and a reduction in NSAID usage. Evidence relating to other fatty acids, antioxidants, zinc, iron, folate, other B vitamins, calcium, vitamin D and fluoride are also considered. The present evidence suggests that RA patients should consume a balanced diet rich in long-chain n-3 PUFA and antioxidants. More randomized long-term studies are needed to provide evidence for the benefits of specific nutritional supplementation and to determine optimum intake, particularly for n-3 PUFA and antioxidants.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2002

In vitro study of fracture strength and marginal adaptation of polyethylene-fibre-reinforced-composite versus glass-fibre-reinforced-composite fixed partial dentures.

This in vitro study was carried out in order to estimate the clinical usability of adhesively luted three-unit posterior fixed partial dentures (FPD) made of a polyethylene-fibre-reinforced-composite system (PFRC) in comparison with a glass-fibre-reinforced-composite system (GFRC). Therefore the fracture strength and marginal adaptation were examined. A total of 16 FPDs of each material combination were manufactured and adhesively luted to human molars. Before and after an artificial ageing process by thermal cycling and mechanical loading (TCML) the quality of the marginal adaptation was examined by evaluating epoxy replicas in a scanning electron microscope (SEM). After TCML the fracture strength of eight FPDs of each series was tested by mechanical loading them to failure. With the remaining eight FPDs the quality of deeper layers of the luting was examined with a dye penetration technique. The calculated median fracture strength values (25/75% percentiles) were 830 N (643/982) for the PFRC and 884 N (684/1,113) for the GFRC. The SEM analysis showed at least 80% of perfect marginal areas for both material combinations in dentine as well as in enamel before and after TCML. Looking at the cement-tooth interfaces the dye penetration technique showed statistically significant better results for the enamel finishing lines than for those in dentine. With finishing lines in dentine the two material combinations showed statistically significant differences for this interface. Fracture force results as well as marginal quality encourage further clinical investigations on both systems although the GFRC performed slightly better than the PFRC.

Cementation↗

PubMed2001

HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas.

HSV-1 inhibits apoptosis of infected cells, presumably to ensure that the infected cell survives long enough to allow completion of viral replication. Because cytotoxic lymphocytes kill their targets via the induction of apoptosis, protection from apoptosis could constitute a mechanism of immune evasion for HSV. Several HSV genes are involved in the inhibition of apoptosis, including Us5, which encodes glycoprotein J (gJ). Viruses deleted for Us5 showed defects in inhibition of caspase activation after Fas ligation or UV irradiation. Transfected cells expressing the Us5 gene product gJ were protected from Fas- or UV-induced apoptosis, as measured by morphology, caspase activation, membrane permeability changes, or mitochondrial transmembrane potential. In contrast, caspase 3 activation in mitochondria-free cell lysates by granzyme (gr)B was inhibited equivalently by Us5 deletion and rescue viruses, suggesting that gJ is not required for HSV to inhibition this process. However, mitochondria-free lysates from transfected cells expressing Us5/gJ were protected from grB-induced caspase activation, suggesting that Us5/gJ is sufficient to inhibit this process. Transfected cells expressing Us5/gJ were also protected from death induced by incubation with purified grB and perforin. These findings suggest that HSV has a comprehensive set of immune evasion functions that antagonize both Fas ligand- and grB-mediated pathways of CTL-induced apoptosis. The understanding of HSV effects on killing by CTL effector mechanisms may shed light on the incomplete control of HSV infections by the immune system and may allow more rational approaches to the development of immune modulatory treatments for HSV infection.

Animals↗

PubMed2001

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure.

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.

Amino Acid Sequence↗

PubMed2001

Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells.

Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4(+)CD25(+) cytotoxic T lymphocyte antigen (CTLA)-4(+) regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4(+)CD25(+) Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4(+) T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4(+) T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4(+) T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.

Abatacept↗

PubMed2001

Biphasic response to human galanin of extracellular acidification in human Bowes melanoma cells.

The metabolic response of galanin GAL1 receptor subtype, endogenously expressed in human Bowes melanoma (HBM) cells, was investigated. Cytosensor microphysiometry was used to determine the extracellular acidification rate. A biphasic response, consisting of a rapid increase in the extracellular acidification rate followed by a decrease below the basal level, was observed after perfusion with human galanin. The magnitude and the rate of onset of both phases were dependent on the galanin concentration. The increase in the extracellular acidification rate (maximum of 25% of basal level; -log(EC(50))=7.23+/-0.14) was transient, whereas the following decrease (maximum of 40% of basal level; -log(EC(50))=7.77+/-0.23) was sustained. The EC(50) values for the increase and decrease were in a similar range. After consecutive galanin administration, the magnitude of the response was the same as for the unexposed cells, indicating the absence of galanin receptor desensitization or internalization in HBM cells. Responses were blocked by pretreatment with pertussis toxin and phorbol-12-myristate-13-acetate (PMA), indicating a G-protein/protein kinase C signalling pathway. Our microphysiometry results show a biphasic response of the extracellular acidification rate mediated by the galanin receptor expressed in HBM cells which has not been described previously for any other endogenously expressed neuropeptide receptor.

1-Methyl-3-isobutylxanthine↗