bluuebunny/crossref_metadata_2025 huggingface dataset:
Gut Vagal Afferents Are Not Necessary for the Eating-Stimulatory Effect of Intraperitoneally Injected Ghrelin in the Rat
Authors: Arnold Myrtha , Mura Anna , Langhans Wolfgang , Geary Nori
Published: October 2006

Ghrelin is unique among gut peptides in that its plasma level increases during fasts and its administration stimulates eating. Although ghrelin physiology has been intensively studied, whether its eating-stimulatory effect arises from endocrine-neural signal transduction at peripheral or central sites remains unresolved. To address this issue, we tested the effects of subdiaphragmatic vagal deafferentation (SDA), the most complete and selective vagal deafferentation method available, on ghrelin-induced eating. SDA was verified with a cholecystokinin satiation test, retrograde labeling of vagal motor neurons in the dorsal motor nucleus of the vagus with fluorogold, and anterograde labeling of vagal afferents in the nucleus tractus solitarius with wheat germ agglutinin-horseradish peroxidase. Intraperitoneal injections of 10–40 μg/kg ghrelin stimulated eating as robustly in rats with verified complete SDA as in sham-operated controls. Ghrelin also stimulated eating in rats with total subdiaphragmatic vagotomies. We also recorded the electrophysiological responses of gastric load-sensitive vagal afferent neurons to intravenous ghrelin. Ghrelin (10 nmol) phasically (0–30 s) increased activity in two of seven gastric load-sensitive fibers in the absence of gastric loads and tonically (5–30 min) increased activity in only one fiber. Ghrelin did not affect any of the eight fibers tested in the presence of 1–3 ml gastric loads. We conclude that although phasic increases in plasma ghrelin may affect the activity of a fraction of gastric load-sensitive vagal afferents, the acute eating-stimulatory effect of intraperitoneal ghrelin does not require vagal afferent signaling.
The Low-Density Lipoprotein Receptor-Related Protein Is a Pro-Survival Receptor in Schwann Cells: Possible Implications in Peripheral Nerve Injury
Authors: Campana W. Marie , Li Xiaoqing , Dragojlovic Nikola , Janes Julie , Gaultier Alban , Gonias Steven L.
Published: October 2006

Schwann cells undergo phenotypic modulation in peripheral nerve injury. In the adult rodent, Schwann cells are resistant to death-promoting challenges. The responsible receptors and signaling pathways are incompletely understood. In this study, we demonstrate that low-density lipoprotein receptor-related protein-1 (LRP-1) is expressed in adult sciatic nerve. After crush injury, LRP-1 is lost from the axoplasm and substantially upregulated in Schwann cells. Increased LRP-1 mRNA expression was observed locally at the injury site in multiple forms of sciatic nerve injury, including crush injury, chronic constriction injury, and axotomy. Endogenously produced tumor necrosis factor-α (TNF-α) was mostly responsible for the increase in LRP-1 expression; this activity was reproduced by direct injection of TNF-α into injured nerves in theTNF-α gene knock-out mouse. TNF receptor II was primarily involved. TNF-α also increased LRP-1 mRNA in Schwann cells in primary culture. Silencing of Schwann cell LRP-1 with siRNA decreased phosphorylated Akt and increased activated caspase-3. Equivalent changes in cell signaling were observed in LRP-1-deficient murine embryonic fibroblasts. Schwann cell death was inducedin vitroby serum withdrawal or TNF-α, to a greater extent when LRP-1 was silenced. Schwann cell death was inducedin vivoby injecting the LRP-1 antagonist, receptor-associated protein, into axotomy sites in adult rats. These results support a model in which LRP-1 functions as a pro-survival receptor in Schwann cells.
Potential New Antiepileptogenic Targets Indicated by Microarray Analysis in a Rat Model for Temporal Lobe Epilepsy
Authors: Gorter Jan A. , van Vliet Erwin A. , Aronica Eleonora , Breit Timo , Rauwerda Han , Lopes da Silva Fernando H. , Wadman Wytse J.
Published: October 2006

To get insight into the mechanisms that may lead to progression of temporal lobe epilepsy, we investigated gene expression during epileptogenesis in the rat. RNA was obtained from three different brain regions [CA3, entorhinal cortex (EC), and cerebellum (CB)] at three different time points after electrically induced status epilepticus (SE): acute phase [group D (1 d)], latent period [group W (1 week)], and chronic epileptic period [group M (3–4 months)]. A group that was stimulated but that had not experienced SE and later epilepsy was also included (group nS). Gene expression analysis was performed using the Affymetrix Gene Chip System (RAE230A). We used GENMAPP and Gene Ontology to identify global biological trends in gene expression data. The immune response was the most prominent process changed during all three phases of epileptogenesis. Synaptic transmission was a downregulated process during the acute and latent phases. GABA receptor subunits involved in tonic inhibition were persistently downregulated. These changes were observed mostly in both CA3 and EC but not in CB. Rats that were stimulated but that did not develop spontaneous seizures later on had also some changes in gene expression, but this was not reflected in a significant change of a biological process. These data suggest that the targeting of specific genes that are involved in these biological processes may be a promising strategy to slow down or prevent the progression of epilepsy. Especially genes related to the immune response, such as complement factors, interleukins, and genes related to prostaglandin synthesis and coagulation pathway may be interesting targets.
Amphetamine-Induced Place Preference and Conditioned Motor Sensitization Requires Activation of Tyrosine Kinase Receptors in the Hippocampus
Authors: Shen Fei , Meredith Gloria E. , Napier T. Celeste
Published: October 2006

The environmental context in which abused drugs are taken contribute to the drug experience and is a powerful and persistent stimulus to elicit memories of that experience even in the abstinent addict. Using amphetamine (AMPH) as the unconditioned stimulus, the present study compared two popular context-dependent paradigms in rats, conditioned motor sensitization (CMS) and conditioned place preference (CPP), to ascertain whether particular brain regions were differentially involved. The neuronal substrates underlying these context-dependent behaviors are poorly understood, but regulators of the neuronal plasticity that accompany learning, such as neurotrophic factors and their cognate tyrosine kinase receptors (e.g., TrkB), are credible candidates. We found a significant elevation of TrkB-like immunoreactivity specifically in CA3/dentate gyrus (DG) subregions of the hippocampus after AMPH (0.3 mg/kg)-induced CPP, but not in the delayed-paired (control) AMPH condition. A higher AMPH dose (1.0 mg/kg) induced both CPP and CMS and elevated TrkB in the CA3/DG as well as in the nucleus accumbens shell. The development of both conditioned behaviors was blocked by intra-CA3/DG infusion of the Trk inhibitor K-252a. These findings reveal that CPP and CMS are induced by different doses of AMPH and are associated with TrkB changes in particular brain regions. Moreover, Trk receptors in the hippocampus are critical mediators of the neuronal changes necessary for inducing both forms of conditioning. Thus, although these two conditioning models are distinct, because they are commonly regulated by the hippocampal Trk system, these receptors may be a therapeutic target for attenuating the significance of contextual cues that otherwise strengthen the addictive properties of abused drugs.
Impaired Inactivation Gate Stabilization Predicts Increased Persistent Current for an Epilepsy-AssociatedSCN1AMutation
Authors: Kahlig Kristopher M. , Misra Sunita N. , George Alfred L.
Published: October 2006

Mutations inSCN1A(encoding the neuronal voltage-gated sodium channel α1subunit, NaV1.1, or SCN1A) are associated with genetic epilepsy syndromes including generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy. Here, we present the formulation and use of a computational model for SCN1A to elucidate molecular mechanisms underlying the increased persistent sodium current exhibited by the GEFS+ mutant R1648H. Our model accurately reproduces all experimentally measured SCN1A whole-cell biophysical properties including biphasic whole-cell current decay, channel activation, and entry into and recovery from fast and slow inactivation. The model predicts that SCN1A open-state inactivation results from a two-step process that can be conceptualized as initial gate closure, followed by recruitment of a mechanism (“latch”) to stabilize the inactivated state. Selective impairment of the second latching step results in an increase in whole-cell persistent current similar to that observed for the GEFS+ mutant R1648H. These results provide a deeper level of understanding of mutant SCN1A dysfunction in an inherited epilepsy syndrome, which will enable more precise computational studies of abnormal neuronal activity in epilepsy and may help guide new targeted therapeutic strategies.
Probabilistic Encoding of Vocalizations in Macaque Ventral Lateral Prefrontal Cortex
Authors: Averbeck Bruno B. , Romanski Lizabeth M.
Published: October 2006

We examined strategies for classifying macaque vocalizations into their corresponding categories, as well as whether or not there was evidence that prefrontal auditory neurons were related to this process. We found that static estimates of the spectral and temporal contrasts of the calls were not effective features for discriminating among the call classes. A hidden Markov model (HMM), however, was more effective at discriminating among the call classes, reaching a performance of almost 75% correct. Finally, we found that the responses of prefrontal auditory neurons could be predicted more effectively as linear functions of the probabilistic output of the HMM than as linear functions of the spectral features of the calls. This provides evidence that, for call recognition, the macaque auditory system likely performs dynamic processing of vocalizations, and that prefrontal auditory neurons carry a signal related to the output of this processing.
Distinct Roles of the β1-Class Integrins at the Developing and the Mature Hippocampal Excitatory Synapse
Authors: Huang Zhen , Shimazu Kazuhiro , Woo Newton H. , Zang Keling , Müller Ulrich , Lu Bai , Reichardt Louis F.
Published: October 2006

Integrins are a large family of cell adhesion receptors involved in a variety of cellular functions. To study their roles at central synapses, we used twocrerecombinase lines to delete theItgb1β1 integrin gene in forebrain excitatory neurons at different developmental stages. Removal of the β1 integrins at an embryonic stage resulted in severe cortical lamination defects without affecting the cellular organization of pyramidal neurons in the CA3 and CA1 regions of the hippocampus. Whereas the hippocampal neurons underwent normal dendritic and synaptic differentiation, the adult synapses exhibited deficits in responses to high-frequency stimulation (HFS), as well as in long-term potentiation (LTP). Deletion of β1 integrin at a later postnatal stage also impaired LTP but not synaptic responses to HFS. Thus, the β1-class integrins appear to play distinct roles at different stages of synaptic development, critical for the proper maturation of readily releasable pool of vesicles during early development but essential for LTP throughout adult life.
The CC genotype of the GNAS T393C polymorphism is associated with obesity and insulin resistance in women with polycystic ovary syndrome
Authors: Hahn Susanne , Frey Ulrich H , Siffert Winfried , Tan Susanne , Mann Klaus , Janssen Onno E
Published: October 2006

Objective: Variants in the Gs protein α subunit gene (GNAS1) are known to be involved in the pathogenesis of several endocrine and metabolic disorders. To understand genetic determinants of androgen excess, insulin resistance, and obesity in polycystic ovary syndrome (PCOS), we investigated the effect of the common GNAS1 T393C polymorphism on the phenotype of German PCOS women.

Design: Two hundred and seventy-eight PCOS women and 820 Caucasian controls were genotyped for the common T393C polymorphism in GNAS1. To this end, genomic DNA was amplified by PCR with specific oligonucleotides and genotypes were determined using the restriction enzyme FokI. In addition, we evaluated whether the T393C polymorphism had an influence on the response to 6 months metformin treatment in a subgroup of 105 PCOS women.

Methods: Anthropometric variables, metabolic parameters including indices of insulin resistance measured by oral glucose tolerance testing, and endocrine biochemical as well as clinical parameters were measured in all PCOS subjects.

Results: GNAS1 genotype distributions were not significantly different between PCOS women and controls. In PCOS women, no significant differences in endocrine clinical and biochemical variables were found between the genotypes. However, the C-allele carrier group had significantly higher mean body weight, body mass index, leptin levels, and higher indices of insulin resistance compared with women with GNAS1 TT-genotype. Metformin treatment significantly improved hyperandrogenism, menstrual cyclicity, body weight, and insulin resistance independent of GNAS1 genotype. The major determinant of weight loss was body weight before treatment.

Conclusion: The T393C polymorphism is not associated with PCOS in Caucasian women, but may represent a genetic marker for increased susceptibility for obesity in this cohort.
Transcription factors regulating β-cell function
Authors: Cerf Marlon E
Published: October 2006

Type 2 diabetes is primarily associated with insulin resistance and β-cell dysfunction. Maintenance of functional mature β-cells is imperative for ensuring glucose homeostasis. This can be achieved by optimal expression of key transcription factors that are required for normal pancreatic development and maintaining β-cell function. Defining the regulation of transcription factors as well as their regulation of important β-cell genes like insulin will provide further insight into elucidating the mechanisms leading to β-cell dysfunction.
Genetic variation in thyroid hormone pathway genes; polymorphisms in the TSH receptor and the iodothyronine deiodinases
Authors: Peeters Robin P , van der Deure Wendy M , Visser Theo J
Published: October 2006

Serum thyroid parameters show substantial inter-individual variability, in which genetic variation is a major factor. Findings in patients with subclinical hyper- and hypothyroidism illustrate that even minor alterations in serum thyroid function tests can have important consequences for a variety of thyroid hormone-related clinical endpoints, such as atherosclerosis, bone mineral density, obesity, and heart rate. In the last few years, several studies described polymorphisms in thyroid hormone pathway genes that alter serum thyroid function tests. In this review, we discuss the genetic variation in the TSH receptor and iodothyronine deiodinases. We discuss the possible consequences of these studies for the individual patient and also the new insights in thyroid hormone action that can be obtained from these data.
Biochemical markers of bone turnover and their relation to forearm bone mineral density in persons of Pakistani and Norwegian background living in Oslo, Norway: The Oslo Health Study
Authors: Holvik Kristin , Meyer Haakon E , Søgaard Anne Johanne , Selmer Randi , Haug Egil , Falch Jan A
Published: October 2006

Objective: To evaluate whether Pakistanis have increased bone turnover compared with ethnic Norwegians due to their high prevalence of vitamin D deficiency and secondary hyperparathyroidism, and whether the relation between bone turnover and bone mineral density (BMD) differs between Pakistanis and ethnic Norwegians.

Design: A cross-sectional, population-based study conducted in the city of Oslo in 2000–2001. Random samples of 132 community-dwelling Pakistani men and women of ages 40, 45, and 59–60 years, and 580 community-dwelling Norwegian men and women of ages 45 and 59–60 years are included in this substudy.

Methods: Venous serum samples were drawn for measurements of markers of the vitamin D endocrine system and the bone turnover markers osteocalcin (s-OC), bone alkaline phosphatase (s-bone ALP), and tartrate-resistant acid phosphatase (s-TRACP). BMD was measured at the forearm by single-energy X-ray absorptiometry.

Results: Pakistanis had higher s-bone ALP compared with Norwegians. Mean (95% CI) age-adjusted levels were 22.5 (21.0, 24.1) U/l in Pakistani men versus 19.3 (18.6, 20.1) U/l in Norwegian men, P < 0.0005, and 20.3 (18.4, 22.1) U/l in Pakistani women versus 16.7 (16.0, 17.4) U/l in Norwegian women, P = 0.001. There tended to be an inverse association between bone turnover and BMD in men and women of both ethnic groups, and it was strongest for s-bone ALP. Overall mean (95% CI) distal BMD decrease was −16 (−20, −11) mg/cm2 per 1 s.d. increase in s-bone ALP (P < 0.0005) when adjusting for age, sex, and ethnicity.

Conclusions: Except for somewhat higher s-bone ALP levels in Pakistanis, there were only minor ethnic differences in bone turnover, despite a strikingly different prevalence of secondary hyperparathyroidism. Bone turnover was inversely associated with forearm BMD in both ethnic groups.
Sustained effects of recombinant GH replacement after 7 years of treatment in adults with GH deficiency
Authors: van der Klaauw A A , Romijn J A , Biermasz N R , Smit J W A , van Doorn J , Dekkers O M , Roelfsema F , Pereira A M
Published: October 2006

Context: The goal of GH replacement with recombinant human GH (rhGH) is to ameliorate symptoms, signs, and complications of adult GH deficiency (GHD) in the long term. To determine whether the observed short-term beneficial effects of rhGH treatment are sustained in the long term, we evaluated biochemical and anthropometric parameters after 7 years of rhGH replacement.

Patients and methods: After 2, 5, and 7 years of rhGH replacement, 63 adult GHD patients (30 men, 52 adult-onset GHD) were assessed. IGF-I increased during rhGH replacement, and a stable dose of rhGH was reached within 1 year of rhGH substitution. Thereafter, this individualized dose was continued.

Results: Plasma levels of total cholesterol and low-density lipoprotein cholesterol decreased even after 5 years of rhGH replacement (11% decrease, P < 0.001; 22% decrease, P < 0.001 respectively). High-density lipoprotein cholesterol levels increased during 7 years of rhGH replacement (1.4 ± 0.5 mmol/l at baseline vs 1.7 ± 0.5 mmol/l after 7 years, P < 0.001), whereas triglyceride concentrations remained unchanged. Fasting glucose levels increased during follow-up, mainly during the first 2 years of rhGH replacement (4.4 ± 0.7 mmol/l to 5.0 ± 1.0 mmol/l, P < 0.001). Body mass index increased during follow-up, whereas waist circumference and waist-to-hip ratio remained unchanged. Diastolic blood pressure decreased (P = 0.002), but when patients using antihypertensive medication were excluded this decrease did not reach significance (P = 0.064). Systolic blood pressure remained unchanged.

Conclusion: The beneficial effects of rhGH replacement, described after short-term rhGH replacement, are sustained in the long term up to 7 years.
Serum GH and IGF-I are significant determinants of bone turnover but not bone mineral density in active acromegaly: a prospective study of more than 70 consecutive patients
Authors: Ueland T , Fougner S L , Godang K , Schreiner T , Bollerslev J
Published: October 2006

Objective: Acromegaly is characterized by a persistent hypersecretion of GH and provides information on long-term effects of GH on bone metabolism. The aim of this study was to examine the effect of gonadal status and disease activity on bone metabolism in active acromegaly.

Methods: Seventy-three consecutive patients with active acromegaly: 40 women and 33 men (50 ± 13 (mean ± s.d.) and 49 ± 10 years respectively) were evaluated and compared with age-, sex-, and body mass index (BMI)-matched controls by X-ray absorptiometry and biochemical analysis (markers of disease activity and bone turnover).

Results: We found that bone turnover, as evaluated by biochemical bone markers, is coupled and markedly increased in relation to disease activity in active acromegaly. Acromegalic women, but not men, were characterized by an increased bone area and slightly decreased bone mineral content resulting in significantly decreased bone mineral density (BMD) in the ultradistal radius, proximal radius, and total body. No differences in bone turnover or BMD were found between eu-and hypogonadal subjects. Multivariate analysis identified age, BMI, and gender as independent predictors of total BMD in acromegaly.

Conclusion: Our study demonstrates a decreased total body BMD in women, not men, with active acromegaly, regardless of gonadal status or disease activity. Bone turnover is markedly increased in relation to disease activity, possibly counteracting the anabolic effects of excess GH/IGF-I in these subjects. We suggest more focus on biomechanical analyses when investigating endocrine disorders affecting bone size and distribution between compartments.
R990G polymorphism of the calcium-sensing receptor and renal calcium excretion in patients with primary hyperparathyroidism
Authors: Corbetta S , Eller-Vainicher C , Filopanti M , Saeli P , Vezzoli G , Arcidiacono T , Loli P , Syren M L , Soldati L , Beck-Peccoz P , Spada A
Published: October 2006

Context: Primary hyperparathyroidism (PHPT) shows a great variability in clinical course and severity. Data concerning the association between polymorphic variants of the gene encoding the calcium-sensing receptor (CaSR) and clinical characteristics of PHPT are not conclusive.

Objective: To evaluate the frequency of three polymorphisms; A986S, R990G, and Q1011E of CaSR in patients with PHPT and to correlate the genotypes with clinical and biochemical parameters.

Patients and methods: The study included 94 consecutive unrelated patients referred to our Departments for PHPT diagnosis and management between 2000 and 2005 and 137 age and sex-matched healthy subjects. Patients and controls were genotyped according to standard procedures. Due to the rarity of 990G allele, homozygous and heterozygous subjects were grouped in R/G+G/G set. All PHPT patients were studied for calcium metabolism parameters and renal and bone complications.

Results: The proportion of CaSRvariants was similar in PHPT patients and controls. In PHPT patients, only R990G polymorphism was associated with disease parameters; in comparison with R/R, R/G+G/G patients showed lower mean serum parathyroid hormone (PTH) and phosphate levels (139.9 ± 62.2 vs 199.9 ± 136.3 pg/ml, P < 0.05 and 0.69 ± 0.12 vs 0.81 ± 0.18 mmol/l, P = 0.031 respectively), higher mean 24-h urine calcium concentration and calcium excretion (9.05 ± 2.05 vs 6.77 ± 4.31 mmol/24 h, P = 0.012 and 67 ± 20 vs 51 ± 26 μmol/l GF, P = 0.039), and increased prevalence of nephrolithiasis (90.0 vs 44.2%, P = 0.007).

Conclusions: The study showed that patients with PHPT, bearing the 990G allele, had lower serum PTH levels and higher urinary calcium excretion in comparison with the other genotype, suggesting an increased sensitivityof the variant receptor to extracellular calcium. Since this variant was associated with increased occurrence of nephrolithiasis, analysis of this polymorphism might help to predict renal complication of the disease.
Low plasma adiponectin concentration is an indicator of the metabolic syndrome
Authors: Santaniemi Merja , Kesäniemi Y Antero , Ukkola Olavi
Published: October 2006

Objective: Adiponectin is an adipocytokine known to be decreased in obesity. It functions in glucose and fatty acid metabolism and also has an anti-inflammatory role in microvasculature. We wanted to investigate the role of adiponectin as a biomarker of metabolic syndrome (MS) and see how the plasma adiponectin levels relate to new criteria of MS proposed by the International Diabetes Federation.

Methods: Plasma adiponectin concentrations were measured from total of 1041 Finnish subjects with an ELISA – a novel method planned in our laboratory.

Results: In both the sexes, the plasma adiponectin levels were lower in subjects with MS when compared with subjects with no diagnosis of MS (P< 0.001). Plasma adiponectin levels did not differ between subjects with National Cholesterol Education Program (ATPIII) and International Diabetes Federation-defined MS. Lower adiponectin levels were associated with different components of the MS and there was a trend towards decreasing adiponectin levels with an increasing number of components of MS in both the sexes. Subjects in the lowest adiponectin quartile had a significantly higher probability of having MS (P< 0.001), 4.4-fold in males and 7.5-fold in females, when compared with the corresponding individuals in the highest quartile. The probability increased in every lowering quartile of adiponectin level and was independent of body mass index.

Conclusion: We conclude that adiponectin levels correlate with most of the components of the MS and the metabolic cluster per se.
Light‐Driven Water Splitting for (Bio‐)Hydrogen Production: Photosystem 2 as the Central Part of a Bioelectrochemical Device
Authors: Badura Adrian , Esper Berndt , Ataka Kenichi , Grunwald Christian , Wöll Christof , Kuhlmann Jürgen , Heberle Joachim , Rögner Matthias
Published: October 2006

To establish a semiartificial device for (bio‐)hydrogen production utilizing photosynthetic water oxidation, we report on the immobilization of a Photosystem 2 on electrode surfaces. For this purpose, an isolated Photosystem 2 with a genetically introduced His tag from the cyanobacterium Thermosynechococcus elongatus was attached onto gold electrodes modified with thiolates bearing terminal Ni(II)‐nitrilotriacetic acid groups. Surface enhanced infrared absorption spectroscopy showed the binding kinetics of Photosystem 2, whereas surface plasmon resonance measurements allowed the amount of protein adsorbed to be quantified. On the basis of these data, the surface coverage was calculated to be 0.29 pmol protein cm−2, which is in agreement with the formation of a monomolecular film on the electrode surface. Upon illumination, the generation of a photocurrent was observed with current densities of up to 14 μA cm−2. This photocurrent is clearly dependent on light quality showing an action spectrum similar to an isolated Photosystem 2. The achieved current densities are equivalent to the highest reported oxygen evolution activities in solution under comparable conditions.
A New Approach for Studying Fast Biological Reactions Involving Nitric Oxide: Generation of NO Using Photolabile Ruthenium and Manganese NO Donors
Authors: Szundi Istvan , Rose Michael J. , Sen Indranll , Eroy‐Reveles Aura A. , Mascharak Pradip K. , Einarsdóttir Ólöf
Published: October 2006

Nitric oxide (NO) is recognized as one of the major players in various biochemical processes, including blood pressure, neurotransmission and immune responses. However, experimental studies involving NO are often limited by difficulties associated with the use of NO gas, including its toxicity and precise control over NO concentration. Moreover, the reactions of NO with biological molecules, which frequently occur on time scales of microseconds or faster, are limited by the millisecond time scale of conventional stopped‐flow techniques. Here we present a new approach for studying rapid biological reactions involving NO. The method is based on designed ruthenium and manganese nitrosyls, [Ru(PaPy3)(NO)](BF4)2 and [Mn(Pa‐Py3)(NO)](ClO4) (PaPy3H =N,N ‐bis(2‐pyridylmethyl)amine‐N‐ethyl‐2‐pyridine‐2‐carboxamide), which upon photolysis produce NO on a fast time scale. The kinetics of the binding of the photogenerated NO to reduced cytochrome c oxidase (CcO) and myoglobin (Mb) was investigated using time‐resolved optical absorption spectroscopy. The NO was found to bind to reduced CcO with an apparent lifetime of 77 μs using the [Mn(PaPy3)(NO)]+ complex; the corresponding rate is 10–20 times faster than can be detected by conventional stopped‐flow methods. Second‐order rate constants of ∼1 × 108 108 M−1 S−1 and ∼3 × 107 M−1 S−1 were determined for NO binding to reduced CcO and Mb, respectively. The generation of NO by photolysis of these complexes circumvents the rate limitation of stopped‐flow techniques and offers a novel alternative to study other fast biological reactions involving NO.
CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP
Authors: Sugawara Teruo , Sakuragi Noriaki , Minakami Hisanori
Published: October 2006

Steroidogenic acute regulatory (StAR) protein plays a critical role in steroid hormone synthesis. Tropic hormones induce human StAR gene expression by a cAMP-dependent pathway. Steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP) plays an important role in the expression of human StAR gene. We investigated the mechanism of cAMP responsiveness in human StAR gene expression in NCI-H295R cells. The StAR promoter activity and protein levels in cells subjected to various treatments were examined. Anti-SF-1/Ad4BP IgG transfection treatment resulted in decreases in the basal StAR promoter activity and StAR protein levels, but did not affect cAMP-stimulated promoter activity and protein levels. The basal and cAMP-stimulated StAR promoter activity levels were reduced in SF-1/Ad4BP mutant (G35E)-transfected cells, but the cAMP induction of StAR promoter activity in response to 1 mM 8-Br-cAMP was not inhibited when G35E SF-1/Ad4BP mutant expression vectors were co-transfected with cAMP-response element-binding (CREB) expression vectors. Although the basal StAR mRNA expression and protein levels were decreased by SF-1/Ad4BP-siRNA treatment, the cAMP-stimulated StAR mRNA expression and protein levels did not change. The basal StAR promoter activity level was not decreased by cAMP-response element modulator (CREM)-siRNA treatment, but the cAMP-stimulated StAR promoter activity level, the magnitude of cAMP induction of StAR promoter, and the cAMP-stimulated StAR protein level were decreased. The cAMP induction of StAR promoter activity in cells was inhibited when S117ACREM mutant expressionvectors were transfected. We conclude that inhibition of the function of SF-1/Ad4BP does not reduce the cAMP induction of StAR promoter activity and protein level. CREM is needed to confer cAMP responsiveness in human StAR protein expression.
Development of an in vitro pancreatic tissue model to study regulation of islet neogenesis associated protein expression
Authors: Petropavlovskaia Maria , Makhlin Julia , Sampalis John , Rosenberg Lawrence
Published: October 2006

Restoration of a functional β-cell mass in a patient with diabetes may hold the key for curing the disease. In recent years, there has been increasing interest in the development of new strategies to induce β-cell regeneration and new islet formation in situ and a role for Reg proteins has been suggested. One such protein, islet neogenesis associated protein (INGAP), is a member of the Reg3 family of proteins and has been shown to induce islet neogenesis. Elucidation of the mechanisms and factors involved in the regulation of expression of INGAP and related proteins is, therefore, of great importance. Here, we report the establishment of the first in vitro tissue model of INGAP expression that consists of epithelial cystic structures derived from hamster pancreatic acinar tissue cultured in collagen matrix. The objective of this study was to characterize INGAP expression in this model and to investigate the role of pro-inflammatory cytokines and growth factors. Using quantitative reverse transcriptase PCR, we show that INGAP expression correlates with cyst formation and size suggesting the involvement of intra-luminal pressure associated with cyst growth. We also demonstrate for the first time that INGAP gene expression was significantly induced by treatment with interleukin (IL)-6 and further enhanced by a combination of IL-6 with dexamethazone and nicotinamide. Additionally, our data suggest that the effect of IL-6 on INGAP expression is mediated via the JAK/STAT3 signaling pathway. In summary, the in vitro model of INGAP expression described here represents an important step in the development of strategies for the use of INGAP and related proteins as islet neogenic agents in the pharmacotherapy of both type-1 and type-2 diabetes.
Effect of prolonged in vitro exposure to high glucose on neonatal porcine pancreatic islets
Authors: Harb George , Korbutt Gregory S
Published: October 2006

Prolonged exposure to high glucose can influence the function, growth, and survival of pancreatic β-cells. In this study, we examine the effects of prolonged in vitro exposure to high glucose on neonatal porcine β-cells, a potentially useful source of insulin-producing cells for clinical islet transplantation. Neonatal porcine islets were prepared by culturing collagenase-digested pancreases for 1 week in 5.6 mM glucose, followed by an additional week in either 5.6, 10.0, or 28.0 mM glucose. An additional 2 days of culture in 5.6 mM glucose followed for recovery from high glucose. The 7-day culture period in 28.0 mM glucose failed to irreversibly impair glucose responsiveness and also caused a modest increase in β-cell mass. Immunostaining revealed that precursor cell differentiation was responsible for the increase in β-cell mass rather than β-cell proliferation. Islet cell survival was also assessed by a DNA fragmentation assay (TUNEL stain) to determine β-cell susceptibility to apoptosis after exposure to high glucose. Interestingly, although the total number of apoptotic islet cells did not drastically change after a week of culture in either 5.6, 10.0, or 28.0 mM glucose (25% TUNEL-positive), neither did the percentage of apoptotic β-cells. These encouraging results further support the use of neonatal porcine islets for clinical transplantation because of their ability to resist the cytotoxic effects of high glucose on islet function and survival.
Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-II
Authors: Pearson Lachlan J , Rait Christopher , Nicholls M Gary , Yandle Timothy G , Evans John J
Published: October 2006

It is well documented that there are gender differences in the incidence and patterns of cardiovascular diseases but the reasons are unclear. Sex steroids may modulate the behaviour of vascular endothelial cells, which in turn act by paracrine processes to alter adjacent vascular smooth muscle activity. We hypothesised that the sex steroids alter the percentage of vascular endothelial cells that secrete the vasodilator peptide, adrenomedullin and modify the adrenomedullin-stimulating action of angiotensin-II. The percentage of adrenomedullin-secreting human aortic endothelial cells was determined using the cell immunoblot method. Cells were incubated with selected concentrations of angiotensin-II, oestradiol and testosterone alone and sex steroids in combination with angiotensin-II. Adrenomedullin mRNA expression in endothelial cells was quantified by real-time PCR. It was observed that at 4 h, angiotensin-II increased the percentage of adrenomedullin-secreting cells in a concentration-dependent manner. Testosterone at physiological concentrations was observed to increase the number of adrenomedullin-secreting cells whilst oestradiol had no effect. Addition of testosterone to angiotensin-II resulted in less than additive increases in the number of cells secreting adrenomedullin. Oestradiol reduced the angiotensin-II-induced increase in adrenomedullin-secreting cells. Adrenomedullin mRNA expression was significantly increased by testosterone and there was also a trend for an increase in adrenomedullin mRNA expression, which occurred when cells were incubated with angiotensin-II. Our results point to a complex interplay between the sex steroids and angiotensin-II in regulating adrenomedullin production by human endothelial cells, which may contribute to gender-related differences in vascular disease in humans.
Effects of peripheral administration of PYY3–36 on feed intake and plasma acyl-ghrelin levels in pigs
Authors: Ito Tetsuya , ThidarMyint Hnin , Murata Takahiro , Inoue Hiroaki , Neyra Roel Mata , Kuwayama Hideto
Published: October 2006

These studies were designed to investigate the effects of i.v. administration of peptide YY3–36 (PYY3–36) on feed intake, acylghrelin, and GH levels in castrated male pigs. Feed intake levels were evaluated during both ad libitum and fast-refed conditions, and plasma hormone responses were evaluated during fasting. During ad libitum feeding, i.v. injection of PYY3–36 (30 μg/kg body weight, BW) significantly reduced feed intake levels within 3 h post-treatment. In the fast-refed condition, both single bolus injection (30 μg/kg BW) and i.v. infusion (0.25 μg/kg BW per min) of PYY3–36 suppressed feed intake levels 1 h post-treatment. Duration of the elevation of plasma PYY levels induced by i.v. injection of porcine PYY3–36 in ad libitum-fed pigs was longer compared with the values of fasted or fast-refed pigs. In the infusion study, the elevation of plasma PYY levels was maintained throughout the infusion period and values were reduced less than half at 15 min after termination of infusion. These results showed that the anorexigenic short-term effect of PYY3–36 treatment corresponds to its half-life. However, i.v. PYY3–36 injection did not influence plasma acyl-ghrelin levels. On the other hand, single bolus injection of PYY3–36 increased plasma GH levels 30 min after treatment. Similar to previous findings in other mammalian species, the results of these studies show that PYY3–36 can reduce feed intake levels; in particular, the effect is potent and acute in pigs. Furthermore, basal plasma PYY levels were higher in ad libitum-fed pigs than in fasted pigs suggesting that circulating PYY3–36 levels influence satiety and contribute to the termination of feed intake in pigs.
Changes in endocrine and neurochemical profiles in neonatal pigs prenatally exposed to increased maternal cortisol
Authors: Kanitz Ellen , Otten Winfried , Tuchscherer Margret
Published: October 2006

Early life environmental factors are able to influence prenatal development and may cause structural and functional effects on hypothalamic–pituitary–adrenal (HPA) axis and neurotransmitter systems in the offspring. These effects seem to be species specific and may depend on the period of gestation when the factors are effective. Elevated maternal cortisol levels are assumed to play a crucial role as a programming factor during prenatal development. Thus, the present study was performed in order to examine the effects of increased maternal cortisol levels during mid- and late gestation on central and peripheral alterations of the HPA axis and brain neurotransmitter profiles in piglets. Endogenous cortisol release was induced by i.m. administration of ACTH to sows every second day either during mid- (day 49 until 75) or late gestation (day 85 until 107). Controls received injections of saline. ACTH treatment of sows during mid- and late gestation had no effects on the gestation length, the number of total born and the frequency of stillborn piglets. However, ACTH treatment during late gestation caused an increase of birth weight (P < 0.04) and affected the organ:body weight ratios (brain and adrenal) in the offspring. There was an impact of increased maternal cortisol on the HPA axis and on central neurotransmitter systems in the offspring. ACTH treatment during mid gestation caused a significant decrease of plasma corticosteroid-binding globulin (CBG; P < 0.03) and an increase of the noradrenergic activity in the locus coeruleus (LC) region (P < 0.02). Elevated maternal cortisol during late gestation also produced a significant decrease of plasma CBG (P < 0.05), but significantly increased the plasma noradrenaline (NA) concentration (P < 0.02) and decreased the serotonergic activity in the LC at both postnatal day 1 (P < 0.016) and day 28 (P < 0.003). Furthermore, there were sex-specific effects of ACTH treatment on plasma CBG, NA and brain monoamine turnover, with more pronounced changes in male offspring. In conclusion, elevated maternal cortisol levels during mid- and late gestation in pigs affect growth, HPA axis and brain neurotransmitter systems in the offspring in a sex-specific manner. The observed alterations in endocrine and neurotransmitter systems are dependent on the gestational period. Late gestation appears to be a more sensitive phase for cortisol-induced programming in pigs. Moreover, the present data show that there are marked developmental differences between laboratory animals and domestic pigs, and highlight the importance of species-specific studies on prenatal influences.
Orexin receptor expression in human adipose tissue: effects of orexin-A and orexin-B
Authors: Digby J E , Chen J , Tang J Y , Lehnert H , Matthews R N , Randeva H S
Published: October 2006

Orexin-A and orexin-B, via their receptors orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R) have been shown to play a role in the regulation of feeding, body weight, and energy expenditure. Adipose tissue also contributes significantly to the maintenance of body weight by interacting with a complex array of bioactive peptides; however, there are no data as yet on the expression of orexin components in adipose tissue. We, therefore, analyzed the expression of OX1R and OX2R in human adipose tissue and determined functional responses to orexin-A and orexin-B. OX1R and OX2R mRNA expression was detected in subcutaneous (s.c.) and omental adipose tissue and in isolated adipocytes. Protein for OX1R and OX2R was also detected in whole adipose tissue sections and lysates. Treatment with orexin-A, and orexin-B (100 nM, 24 h) resulted in a significant increase in peroxisome proliferator-activated receptors γ-2 mRNA expression in s.c. adipose tissue (P < 0.05). Hormone sensitive lipase mRNA was significantly reduced in omental adipose tissue with orexin-A and orexin-B treatment (P < 0.05). Glycerol release from omental adipose tissue was also significantly reduced with orexin-A treatment (P < 0.05).

These findings demonstrate for the first time the presence of functional orexin receptors in human adipose tissue and suggest a role for orexins in adipose tissue metabolism and adipogenesis.
Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals
Authors: Dinan Laurence , Lafont René
Published: October 2006

Zooecdysteroids (arthropod steroid hormones) regulate the development of arthropods and probably many other invertebrates. Phytoecdysteroids are analogues occurring in a wide range of plant species, where they contribute to the deterrence of phytophagous invertebrates. The purpose of this short review is to summarise findings on the occurrence, metabolism and pharmacological effects of ecdysteroids in mammalian systems and to draw attention to their potential applications, particularly in gene-switch technology, where ecdysteroid analogues (steroidal and non-steroidal) can be used as effective and potent elicitors.
Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3–42) and GLP-1(9–36)amide, on insulin secretion and glucose homeostasis in ob/ob mice
Authors: Parker J C , Lavery K S , Irwin N , Green B D , Greer B , Harriott P , O’Harte F P M , Gault V A , Flatt P R
Published: October 2006

Glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3–42) and GLP-1(9–36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9–36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3–42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3–42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.
Increased apoptosis of lactotrophs in streptozotocin-induced diabetic rats is followed by increased proliferation
Authors: Arroba Ana I , Lechuga-Sancho Alfonso M , Frago Laura M , Argente Jesús , Chowen Julie A
Published: October 2006

Poorly controlled diabetes mellitus can result in decreased prolactin production and thus problems with lactation, reproduction, and other physiological processes. This may be due to a loss of lactotrophs, as we have previously shown that long-term (8 weeks) poorly controlled streptozotocin-induced diabetes results in increased death of lactotrophs and that this most likely occurs through the activation of caspase-8 and the extrinsic cell death cascade. However, cell proliferation is also increased in the anterior pituitary at this time, although the cell type undergoing this proliferation and whether it is a response to the increased cell death remains unknown. In order to determine the time-course of increased cell death and proliferation in the anterior pituitary and if this is related to changes in tumor necrosis factor (TNF)-α, a cytokine involved in the activation of the extrinsic cell death pathway, rats were killed at 1, 4, 6, and 8 weeks after the induction of diabetes. Cell death was significantly increased after 4 weeks, as was caspase-8 activation, although circulating levels of TNF-α were increased as early as 1 week. Pituitary levels of TNF-α did not change significantly until 8 weeks after diabetes onset. Similarly, Western-blot analysis of proliferating cell nuclear antigen showed that anterior pituitary cell proliferation increased significantly 8 weeks after diabetes onset, with the majority of proliferating cells, as detected by BrdU incorporation, corresponding to lactotrophs. These results suggest that the increased death of lactotrophs in poorly controlled diabetic rats is followed by increased proliferation of this cell type, even when no treatment is given.