Biomedical subjects

P Barrett

Publications and source records attributed to P Barrett.

At least 19 recordsLinked to original sources

PubMed2007

G protein-coupled receptor 101 mRNA expression in the mouse brain: altered expression in the posterior hypothalamus and amygdala by energetic challenges.

GPCR101 is a recently identified orphan G protein-coupled receptor (GPCR) expressed abundantly in the human and mouse hypothalamus. In the absence of a ligand, a direct approach to determine the function(s) of this receptor is not possible. However, clues to the possible functions of GPCR101 may yield from information on the distribution of the receptor and the effect of in vivo manipulation upon the expression level of the receptor. In situ hybridisation on mouse brain sections revealed GPCR101 expression in a number of nuclei, including the amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, as well as in the arcuate nucleus, posterior hypothalamus and paraventricular nucleus of the hypothalamus. Food-deprivation was found to increase GPCR101 mRNA level in the posterior hypothalamus and amygdala. In obese mice bearing the ob gene mutation, GPCR101 mRNA level decreased in the posterior hypothalamus and remained unaltered in the amygdala. By contrast, in both nuclei, GPCR101 mRNA level did not change significantly in obese ob/ob mice after intraperitoneal injection of leptin or in mice fed with a high fat diet. These data suggest that GPCR101 mRNA expression in the posterior hypothalamus and amygdala is regulated by a factor(s) other than leptin. Dual in situ hybridisation was used to establish the relationship between GPCR101 and neuropeptides expressed in the hypothalamus. In the arcuate nucleus, GPCR101 mRNA was expressed in approximately half of the population of neurones expressing the mRNA for the anorexigenic neuropeptide, pro-opiomelanocortin, which suggests a potential functional relationship.

Amygdala↗

PubMed2006

Regulation of MT melatonin receptor expression in the foetal rat pituitary.

During development, melatonin receptors are transiently expressed in multiple neuroendocrine tissues, suggesting a novel role for melatonin in developmental physiology. The best characterised model of melatonin signalling during development is the pars distalis of the rat pituitary. However, although many studies have characterised the postnatal decline of melatonin receptors in the rat pars distalis, the mechanism(s) that time the developmental onset of receptor expression during embryogenesis are unknown. Analysis of these mechanisms may yield important information regarding the putative role of melatonin in neuroendocrine development. Here, we report the expression of MT(1) melatonin receptor mRNA in the rat pituitary from embryonic day 15.5 (e15.5). Prior to e15.5, the homeodomain transcription factor Msx-1, an inhibitor of cellular differentiation, is widely expressed throughout the pituitary. In transient transfection experiments, Msx-1 potently inhibited pituitary homeobox-1 (Pitx-1)-induced MT(1) promoter activity and therefore may represent a key inhibitor of MT(1) expression in early pituitary development. During late embryogenesis, MT(1) mRNA was expressed in both the ventral and dorsal pituitary. Analysis of a 1.5-kb fragment of the rat MT(1) promoter revealed four putative cis-elements for the POU domain factor Pit-1, which is associated with mid-dorsal cell lineages. Although Pit-1 induced a strong, dose-dependent stimulation of MT(1) promoter activity in vitro, dual-labelled in situ hybridisation revealed no colocalisation of MT(1) and Pit-1 mRNAs in vivo at e19.5. By contrast, all MT(1) positive cells colocalised with alphaGSU and most with betaTSH mRNA. Our data therefore implicate the decline of Msx-1 expression as a key event that times the onset of melatonin receptor expression to the differentiation of endocrine cells types in the developing pituitary gland, and suggest that the melatonin-sensitive cells in the embryonic pituitary are primarily Pit-1-independent thyrotrophs in the rostral pituitary, with a secondary population of pars distalis gonadotrophs.

Animals↗

PubMed2006

The suppressor of cytokine signalling 3, SOCS3, may be one critical modulator of seasonal body weight changes in the Siberian hamster, Phodopus sungorus.

The Siberian hamster, Phodopus sungorus, exhibits a remarkable cycle of body weight, reproduction and leptin sensitivity in response to a seasonal change in photoperiod. In the present study, we investigated the hypothesis that the suppressor of cytokine signalling 3 (SOCS3) plays a critical role in the regulation of the seasonal body weight cycle. We analysed arcuate nucleus SOCS3 gene expression in short day length (SD; 8 : 16 h light/dark) acclimated Siberian hamsters that were transferred back to long day length (LD; 16 : 8 h light/dark) and in hamsters that spontaneously became photorefractory to SD induced by prolonged exposure. SD acclimated hamsters that were transferred back to LD for 1, 2, 3, 4 or 6 weeks, increased arcuate nucleus SOCS3 gene expression to the LD level within 2 weeks, and maintained this higher level thereafter. The early increase of SOCS3 gene expression preceded the LD-induced rise in body weight by approximately 3 weeks. Hamsters kept in SD for an extended period (25 weeks), began to become refractory to SD and to increase body weight. By this time, there was no difference in level of SOCS3 gene expression between LD and SD photoperiods, although body weight was still suppressed in SD hamsters. Finally, we addressed whether SOCS3 gene expression is related to SD-induced gonadal regression or to body weight decrease by comparing Siberian hamsters with Syrian hamsters. The latter exhibited substantial SD-induced gonadal regression but only limited seasonal changes in body weight. Acclimation to either LD or SD for 14 weeks had no effect on SOCS3 gene expression. This implies that arcuate nucleus SOCS3 gene expression is unlikely to be related to seasonal cycles in reproductive activity. Taken together, the findings further strengthen our hypothesis that SOCS3 may be one molecular trigger of seasonal cycles in body weight.

Acclimatization↗

PubMed2006

PC1/3 and PC2 gene expression and post-translational endoproteolytic pro-opiomelanocortin processing is regulated by photoperiod in the seasonal Siberian hamster (Phodopus sungorus).

A remarkable feature of the seasonal adaptation displayed by the Siberian hamster (Phodopus sungorus) is the ability to decrease food intake and body weight (by up to 40%) in response to shortening photoperiod. The regulating neuroendocrine systems involved in this adaptation and their neuroanatomical and molecular bases are poorly understood. We investigated the effect of photoperiod on the expression of prohormone convertases 1 (PC1/3) and 2 (PC2) and the endoproteolytic processing of the neuropeptide precursor pro-opiomelanocortin (POMC) within key energy balance regulating centres of the hypothalamus. We compared mRNA levels and protein distribution of PC1/3, PC2, POMC, adrenocorticotrophic hormone (ACTH), alpha-melanocyte-stimulating hormone (MSH), beta-endorphin and orexin-A in selected hypothalamic areas of long day (LD, 16:8 h light:dark), short day (SD, 8:16 h light:dark) and natural-day (ND, photoperiod depending on time of the year) acclimated Siberian hamsters. The gene expression of PC2 was significantly higher within the arcuate nucleus (ARC, P < 0.01) in SD and in ND (versus LD), and is reflected in the day length profile between October and April in the latter. PC1/3 gene expression in the ARC and lateral hypothalamus was higher in ND but not in SD compared to the respective LD controls. The immunoreactivity of PC1/3 cleaved neuropeptide ACTH in the ARC and PC1/3-colocalised orexin-A in the lateral hypothalamus were not affected by photoperiod changes. However, increased levels of PC2 mRNA and protein were associated with higher abundance of the mature neuropeptides alpha-MSH and beta-endorphin (P < 0.01) in SD. This study provides a possible explanation for previous paradoxical findings showing lower food intake in SD associated with decreased POMC mRNA levels. Our results suggest that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by post-translational processing mediated by the prohormone convertases PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors.

Adaptation, Physiological↗

PubMed2006

'Let a hundred flowers blossom, let a hundred schools of thought contend': a case for therapeutic pluralism in mental health nursing.

With the dominance of cognitive behavioural therapies within mental health nursing educational and practice settings, a danger exists that a narrow application of therapy interventions and micro skills will result. Given the rich and diverse variations of presenting circumstances to community mental health services, an incorporation of therapy approaches beyond the cognitive behavioural model is essential. This paper initially aims to demonstrate the core values of mental health nursing as being at least partially incongruent with those of cognitive behavioural therapies. This paper also aims to provide brief examples of the use of alternate evidential practice-based therapies more reflective of mental health nursing values across a spectrum of community mental health service where the authors are employed.

Community Mental Health Services↗

PubMed2005

How good is zoom endoscopy for assessment of villous atrophy in coeliac disease?

BACKGROUND AND STUDY AIMS: The advent of magnification endoscopy may allow the macroscopic detection of unrecognised villous atrophy in patients with unsuspected coeliac disease. In addition, it may also be possible to use this method to assess the degree of villous atrophy. The aim of this study was to determine the accuracy of zoom endoscopy for the macroscopic evaluation of villous atrophy, in comparison with histological evaluation. PATIENTS AND METHODS: The zoom endoscope provided a magnification capability of x 115. A scoring system (Z score) was devised for grading the appearances of villous atrophy: "Z1" for normal mucosa, "Z2" for stunted villi, "Z3" for markedly stunted villi (with ridges and pits) and "Z4" for a flat mucosa. A total of 53 consecutive patients with treated coeliac disease were followed up over almost 2 years using the Olympus GIF-Q240Z zoom endoscope; a total of 80 procedures were carried out. Four biopsies from the second part of the duodenum were taken from each patient for histological assessment. Histological assessment of villous atrophy was made by a pathologist blinded to the Z score. The correlation between the Z score and the histological score was assessed using the weighted kappa method. RESULTS: The kappa score for the correlation between the macroscopic assessment of villous atrophy and the histology was 0.631, indicating fair to good reproducibility. Agresti's method revealed a very strong baseline association between the two methods ( P < 0.001). Zoom endoscopy had a positive predictive value of 83 % and a negative predictive value of 77 % in detecting villous atrophy. CONCLUSIONS: Our findings suggest that zoom endoscopy may be valuable in assessing the degree of villous atrophy. However, further studies are needed to assess its efficacy in routine practice as a screening or case-finding tool.

Adult↗

PubMed2005

The human MT1 melatonin receptor stimulates cAMP production in the human neuroblastoma cell line SH-SY5Y cells via a calcium-calmodulin signal transduction pathway.

Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2005

Hypothalamic energy balance gene responses in the Sprague-Dawley rat to supplementation of high-energy diet with liquid ensure and subsequent transfer to chow.

Energy dense, high fat, high sugar, foods and beverages in our diet are a major contributor to the escalating global obesity problem. Here, we examine the physiological and neuroendocrine effects of feeding rats a solid high-energy (HE) diet with or without a liquid supplement (Ensure) and the consequence of subsequently transferring animals back to chow (C). Outbred Sprague-Dawley rats were fed C until 49-56 days of age, and then transferred a HE diet for 3 weeks before allocation to one of two weight-matched groups. Over the next 10 weeks, one group remained on HE diet, whereas the other had access to the liquid diet, chocolate Ensure (EN), in addition to HE diet (HE + EN). Half the rats from each group were then killed, and the remainder were returned to C for 3 weeks. Supplementation of the HE diet with EN accelerated weight gain and increased daily energy intake, adipose tissue mass, and circulating leptin levels. Transferring animals back to C caused a decrease in bodyweight in the HE + EN group, whereas HE animals were weight stable. Both groups also exhibited voluntary hypophagia, although the magnitude and duration of this response was greater in HE + EN animals. The only effect of Ensure on the hypothalamic genes studied was on tyrosine kinase B expression in the ventromedial hypothalamic nucleus (VMH), which was increased in rats given the supplement. Withdrawal of the obesogenic diets decreased gene expression for cocaine-and-amphetamine regulated transcript (CART) and dynorphin (DYN) in the arcuate nucleus (ARC), and DYN and brain-derived neurotrophic factor (BDNF) in the VMH, whereas neuropeptide Y (NPY) gene expression in the ARC was increased. These changes were independent of previous dietary history. EN supplementation generates distinct physiological responses, yet has a minimal effect on hypothalamic neuropeptide or receptor gene expression, possibly due to the development of leptin resistance. Withdrawal of obesogenic diets induces changes in the gene expression consistent with NPY, CART and BDNF attempting to oppose weight gain on either HE or HE + EN.

Animals↗

PubMed2003

Lamotrigine for HIV-associated painful sensory neuropathies: a placebo-controlled trial.

OBJECTIVE: To evaluate the efficacy and tolerability of lamotrigine (LTG) for the treatment of pain in HIV-associated sensory neuropathies. METHODS: In a randomized, double-blind study, patients with HIV-associated distal sensory polyneuropathy (DSP) received LTG or placebo during a 7-week dose escalation phase followed by a 4-week maintenance phase. Randomization was stratified according to whether or not patients were currently using neurotoxic antiretroviral therapy (ART). RESULTS: The number of patients randomized was 92 (62 LTG, 30 placebo) in the stratum receiving neurotoxic ART and 135 (88 LTG, 47 placebo) in the stratum not receiving neurotoxic ART. Mean change from baseline in Gracely Pain Scale score for average pain was not different between LTG and placebo at the end of the maintenance phase in either stratum, but the slope of the change in Gracely Pain Scale score for average pain reflected greater improvement with LTG than with placebo in the stratum receiving neurotoxic ART (p = 0.004), as did the mean change from baseline scores on the Visual Analogue Scale for Pain Intensity and the McGill Pain Assessment Scale and patient and clinician ratings of global impression of change in pain (p </= 0.02). The incidence of adverse events, including rash, was similar between LTG and placebo. CONCLUSIONS: Lamotrigine was well-tolerated and effective for HIV-associated neuropathic pain in patients receiving neurotoxic antiretroviral therapy. Additional research is warranted to understand the differing response among patients receiving neurotoxic antiretroviral therapy compared with those not receiving neurotoxic antiretroviral therapy.

Adult↗

PubMed2003

Markers of experimental acute inflammation in the Wistar Han rat with particular reference to haptoglobin and C-reactive protein.

C-reactive protein (CRP), haptoglobin (Hp) and fibrinogen (Fbgn) are acute phase reactants (APRs), the blood levels of which increase during acute inflammation. However, although the levels of these APRs are used to monitor inflammation in man, their usefulness and sensitivity as markers of inflammation in rodents are less clear. We therefore wished to evaluate, in a comparative fashion, a prototype immunoassay for serum CRP, a commercial assay for serum Hp, and an automated assay for Fbgn, using a model of acute inflammation in the rat. Additionally, pro-inflammatory cytokines and serum protein fractions were also measured. The model of inflammation used was the intraperitoneal injection of Freund's complete adjuvant (FCA). In a concluding experiment, findings with Hp in the FCA rat model were validated in a toxicologically relevant study involving the induction of acute hepatic inflammation using the model hepatotoxicant carbon tetrachloride (CCl(4)). Female Wistar Han rats were treated with a single injection of FCA in a dose-response study (1.25-10.0 ml/kg, sampling at 36 h) and two time-course studies (over 40 h and 21 days). In a final experiment, rats were dosed with CCl(4) at 0.8 ml/kg and sampled over a 17-day period. In FCA and CCl(4) experiments, serum/plasma was prepared and tissues taken at autopsy for histological assessment (CCl(4) study only). In the dose-response study, serum CRP, Hp and plasma Fbgn were increased at all FCA dose levels at 36 h post-dosing. Serum alpha(2) and beta(1) globulin fractions were also increased, while albumin levels were decreased. In the 40-h time-course study, CRP levels peaked at 25-40 h post-dosing, to approximately 120% of control (as 100%). Hp levels increased to a maximum at 25 and 40 h post-dosing with values greater than 400% of control, and alpha(2) and beta(1) globulin fractions peaked at 30 and 40 h post-dosing to 221 and 187% of control, respectively. Increased serum interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) levels peaked at 20 h (11-fold) and 25 h (19-fold), respectively. In a 21-day time-course study, no increased CRP levels were measured despite elevated levels of Hp, which peaked at 36 h (approximately 7-fold above control), and remained elevated up to 21 days. IL-6 and IL-1beta levels peaked at 12 h (19-fold) and 24 h (28-fold), respectively. Liver histopathology of animals treated with CCl(4) showed centrilobular hepatocellular degeneration and necrosis (most significant at 36 h) with an inflammatory response (most significant at 48 h). Resolution of the lesion was complete by 4 days post-dosing. Serum alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase levels peaked at 36 h post-dosing. Hp levels increased maximally at 48 h (426% of control). We conclude that serum CRP is a poor marker of acute inflammation in the rat in comparison with serum Hp and plasma Fbgn. Between Hp and Fbgn, serum Hp is shown to be the most sensitive and useful marker of acute inflammation.

Acute Disease↗

PubMed2003

Precursor-protein convertase 1 gene expression in the mouse hypothalamus: differential regulation by ob gene mutation, energy deficit and administration of leptin, and coexpression with prepro-orexin.

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.

Animals↗

PubMed2003

Melatonin action in the pituitary: neuroendocrine synchronizer and developmental modulator?

Melatonin inhibits the gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone and follicle-stimulating hormone from the pars distalis region of the neonatal rat pituitary gland. Over the initial weeks of postnatal life, this response to melatonin declines in parallel with a loss of iodo-melatonin binding sites. Although neonatal gonadotrophs have since been extensively used to study melatonin receptor signalling pathways, the mechanisms driving this phenomenon, together with its physiological significance, remain unknown. Melatonin receptors are expressed in the foetal pars distalis before activation of the GnRH system. Furthermore, the MT1 melatonin receptor promoter contains response elements for transcription factors involved in both pituitary differentiation and gonadotroph regulation. These data, coupled with the known ability of melatonin to regulate rhythmical gene expression in adult pars tuberalis cells, leads us to propose that melatonin acts in the developing animal as a regulator of internal synchrony between tissues.

Animals↗

PubMed2003

Sensitization: a mechanism for melatonin action in the pars tuberalis.

Sensitization of adenylate cyclase is a recently discovered phenomenon. Melatonin can induce a sensitized response of adenylate cyclase in ovine pars tuberalis cells where the receptor for melatonin is endogenously expressed. Although the mechanism is not fully understood, sensitization of adenylate cyclase may be an important part of the mechanism by which melatonin encodes daylength in the pars tuberalis of sheep and other animals. We used this as a hypothesis to search for a natural ligand that would activate adenylate cyclase in ovine pars tuberalis cells. The approach revealed pituitary adenylate cyclase activating polypeptide to be an indirect activator of adenylate cyclase in the ovine pars tuberalis. We discuss this in relation to the mechanism and importance of sensitization to the function to the pars tuberalis.

Adenylyl Cyclases↗

PubMed2003

Neuromedin U and Neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status.

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

Animals↗

PubMed2003

Photoperiodic programming of body weight through the neuroendocrine hypothalamus.

The photoperiodic mammal undergoes quite remarkable changes in physiology as part of its natural adaptations to seasonal fluctuations in the environment. Changes in energy balance and body weight are among these adaptations. In some seasonal mammals, such as the Siberian hamster (Phodopus sungorus), these changes in body weight have been explored in detail, and there is evidence for tightly controlled systems of energy balance that are coordinated by photoperiod acting via the temporal pattern of melatonin secretion from the pineal gland. The pathways and systems involved appear to be quite distinct from the hypothalamic pathways identified to regulate energy balance in studies of both mice and rats thus far. Instead it appears that in the Siberian hamster a tightly regulated system under the control of photoperiod is able to reset the tone of the systems involved in energy balance regulation. Understanding how photoperiod and melatonin act within the hypothalamus to regulate energy balance offers potentially fundamental and important new insights into the control of energy balance. This review describes the current state of our knowledge.

Animals↗

PubMed2002

HPA axis response to a psychological stressor in generalised social phobia.

Social phobia may be associated with a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis. In this study we determined HPA axis responsivity to a psychological stressor in patients with social phobia and compared them to healthy controls. Fifteen patients with DSM IV social phobia with a mean score of 77.7 on the Liebowitz Social Anxiety Scale and 15 age and sex matched controls underwent the stressor consisting of mental arithmetic and a short term memory test performed in front of an audience. Plasma levels of cortisol and corticotropin were measured at various intervals throughout the test. Although baseline measures of cortisol did not differ between patients (319.8+/-34.6 nmol/l) and controls (279.5+/-42.7 nmol/l)(t=0.7, df=28, P<0.5) nor did baseline corticotropin values (8.6+/-2.1 pg/ml vs 13.7+/-2.0 pg/ml respectively) (t=-1.8, df=28, P<0.08) this stressor resulted in a significantly greater delta max cortisol response (the difference between baseline values and the maximum increase during the stressor) in patients (167.1+/-23.7 nmol/l) than in controls (106.7+/-16 nmol/l) (t=2.1, df=28, P<0.04). There was no significant difference in delta max corticotropin between groups (patients 8.8+/-2.1 pg/ml vs controls 9.1+/-1.9 pg/ml) (t=-0.08, df=28, P<0.9). This preliminary study indicates that patients with social phobia appear to have a hyper-responsive adrenocortical response to psychological stress.

Adrenal Glands↗

PubMed2001

Induction of cytotoxic T-cell responses against immunoglobulin V region-derived peptides modified at human leukocyte antigen-A2 binding residues.

Cytotoxic T-lymphocyte (CTL) responses can be generated against peptides derived from the immunoglobulin (Ig) V region in some but not all patients. The main reason for this appears to be the low peptide-binding affinity of Ig-derived peptides to major histocompatibility complex (MHC) class I molecules and their resulting low immunogenicity. This might be improved by conservative amino acid modifications at the MHC-binding residues of the peptides (heteroclitic peptides). In this study, it was found that in 18 Ig-derived peptides, that heteroclitic peptides from the Ig gene with improved binding to human leukocyte antigen (HLA)-A*0201 can be used to improve CTL responses. Amino acid substitution substantially increased predicted binding affinity, and there was a strong correlation between predicted and actual binding to HLA-A*0201. CTLs generated against the heteroclitic peptide had not only enhanced cytotoxicity against the heteroclitic peptide but also increased killing of antigen-presenting cells pulsed with the native peptide. Surprisingly, no difference was observed in the frequency of T cells detected by MHC class I peptide tetramers after stimulation with the heteroclitic peptide compared with the native peptide. CTLs generated against heteroclitic peptides could kill patients' tumor cells, showing that Ig-derived peptides can be presented by the tumor cell and that the failure to mount an immune response (among other reasons) likely results from the low immunogenicity of the native Ig-derived peptide. These results suggest that heteroclitic Ig-derived peptides can enhance immunogenicity, thereby eliciting immune responses, and that they might be useful tools for enhancing immunotherapy approaches to treating B-cell malignant diseases.

Amino Acid Sequence↗

PubMed2001

Serine residues 110 and 114 are required for agonist binding but not antagonist binding to the melatonin MT(1) receptor.

Site-directed mutation of serine 110 (Ser(3.35)) and serine 114 (Ser(3.39)) in the human melatonin MT(1) receptor to alanine residues reduced ligand binding affinities of seven known melatonin receptor agonists and partial agonists by 3- to 15-fold. These mutants also displayed a relative reduction in their affinities for melatonin-mediated functional responses of 30- and 14-fold, respectively. In contrast to the observed effects of the agonists and partial agonists, the melatonin receptor antagonist luzindole was found to bind to mutants Ser(3.35)Ala and Ser(3.39)Ala with affinities equivalent to that determined for the wild-type melatonin MT(1) receptor. Luzindole was subsequently confirmed as an antagonist of melatonin-mediated functional responses for both mutant receptors. These studies have identified that in the human melatonin MT(1) receptor, Ser(3.35) and Ser(3.39), in transmembrane domain 3, are critical for the formation of the high-affinity ligand binding site for agonists and partial agonists but not for the antagonist luzindole.

Acetamides↗