bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid screening of Salmonella entericaserovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles
Authors: Hong Yang , Liu Tongrui , Lee Margie D , Hofacre Charles L , Maier Marie , White David G , Ayers Sherry , Wang Lihua , Berghaus Roy , Maurer John J
Published: October 2008

Background

Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.

Results

By analyzing the nucleotide sequences of the genes for O-antigen biosynthesis including wba operon and the central variable regions of the H1 and H2 flagellin genes in Salmonella, designated PCR primers for four multiplex PCR reactions were used to detect and differentiate Salmonella serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z10; and H2 antigen complexes, I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z15. Through the detection of these antigen gene allele combinations, we were able to distinguish among S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. The assays were useful in identifying Salmonella with O and H antigen gene alleles representing 43 distinct serovars. While the H2 multiplex could discriminate between unrelated H2 antigens, the PCR could not discern differences within the antigen complexes, 1,2; 1,5; 1,6; 1,7 or e,n,x; e,n,z15, requiring a final confirmatory PCR test in the final serovar reporting of S. enterica.

Conclusion

Multiplex PCR assays for detecting specific O and H antigen gene alleles can be a rapid and cost-effective alternative approach to classical serotyping for presumptive identification of S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Reducing unnecessary prescriptions of antibiotics for acute cough: Adaptation of a leaflet aimed at Turkish immigrants in Germany
Authors: Sahlan Selime , Wollny Anja , Brockmann Silke , Fuchs Angela , Altiner Attila
Published: October 2008

Background

The reduction in the number of unnecessary prescriptions of antibiotics has become one of the most important objectives for primary health care. German GPs report that they are under "pressure to prescribe" antibiotics particularly in consultations with Turkish immigrants. And so a qualitative approach was used to learn more about the socio-medical context of Turkish patients in regard to acute coughs. A German leaflet designed to improve the doctor-patient communication has been positively tested and then adapted for Turkish patients.

Methods

The original leaflet was first translated into Turkish. Then 57 patients belonging to 8 different GPs were interviewed about the leaflet using a semi-standardised script. The material was audio recorded, fully transcribed, and analysed by three independent researchers. As a first step a comprehensive content analysis was performed. Secondly, elements crucial to any Turkish version of the leaflet were identified.

Results

The interviews showed that the leaflets' messages were clearly understood by all patients irrespective of age, gender, and educational background. We identified no major problems in the perception of the translated leaflet but identified several minor points which could be improved. We found that patients were starting to reconsider their attitudes after reading the leaflet.

Conclusion

The leaflet successfully imparted relevant and new information to the target patients. A qualitative approach is a feasible way to prove general acceptance and provides additional information for its adaptation to medico-cultural factors.
Do the malnutrition universal screening tool (MUST) and Birmingham nutrition risk (BNR) score predict mortality in older hospitalised patients?
Authors: Henderson Sarah , Moore Nicola , Lee Emma , Witham Miles D
Published: October 2008

Background

Undernutrition is common in older hospitalised patients, and routine screening is advocated. It is unclear whether screening tools such as the Birmingham Nutrition Risk (BNR) score and the Malnutrition Universal Screening Tool (MUST) can successfully predict outcome in this patient group.

Methods

Consecutive admissions to Medicine for the Elderly assessment wards in Dundee were assessed between mid-October 2003 and mid-January 2004. Body Mass Index (BMI), MUST and BNR scores were prospectively collected. Time to death was obtained from the Scottish Death Register and compared across strata of risk.

Results

115 patients were analysed, mean age 82.1 years. 39/115 (34%) were male. 20 patients were identified as high risk by both methods of screening. A further 10 were categorised high risk only with the Birmingham classification and 12 only with MUST.

80/115 (67%) patients had died at the time of accessing death records. MUST category significantly predicted death (log rank test, p = 0.022). Neither BMI (log rank p = 0.37) or Birmingham nutrition score (log rank p = 0.35) predicted death.

Conclusion

The MUST score, but not the BNR, is able to predict increased mortality in older hospitalised patients.
Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures
Authors: Singh Shweta , Gras Adrien , Fiez-Vandal Cédric , Ruprecht Jonathan , Rana Rohini , Martinez Magdalena , Strange Philip G , Wagner Renaud , Byrne Bernadette
Published: October 2008

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.
Persistent left superior vena cava: a case report and review of literature
Authors: Goyal Sandeep K , Punnam Sujeeth R , Verma Gita , Ruberg Frederick L
Published: October 2008

Persistent left superior vena cava is rare but important congenital vascular anomaly. It results when the left superior cardinal vein caudal to the innominate vein fails to regress. It is most commonly observed in isolation but can be associated with other cardiovascular abnormalities including atrial septal defect, bicuspid aortic valve, coarctation of aorta, coronary sinus ostial atresia, and cor triatriatum. The presence of PLSVC can render access to the right side of heart challenging via the left subclavian approach, which is a common site of access utilized when placing pacemakers and Swan-Ganz catheters. Incidental notation of a dilated coronary sinus on echocardiography should raise the suspicion of PLSVC. The diagnosis should be confirmed by saline contrast echocardiography.
Adenocarcinoma of the third portion of the duodenum in a man with CREST syndrome
Authors: Anastasopoulos Georgios , Marinis Athanasios , Konstantinidis Christos , Theodosopoulos Theodosios , Fragulidis Georgios , Vassiliou Ioannis
Published: October 2008

Background

CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly and Telangiectasias) syndrome has been rarely associated with other malignancies (lung, esophagus).This is the first report of a primary adenocarcinoma of the third portion of the duodenum in a patient with CREST syndrome.

Case presentation

A 54-year-old male patient with CREST syndrome presented with colicky postprandial pain of the upper abdomen, diminished food uptake and a 6-Kg-body weight loss during the previous 2 months. An ulcerative lesion in the third portion of the duodenum was revealed during duodenoscopy, with a diagnosis of adenocarcinoma on biopsy specimen histology. The patient underwent a partial pancreatoduodenectomy. No adjuvant therapy was instituted and follow-up is negative for local recurrence or metastases 21 months postoperatively.

Conclusion

CREST syndrome has been associated with colon cancer, gastric polyps, familial adenomatous polyposis (FAP) syndrome and Crohn's disease; however, this is the first report of a primary adenocarcinoma of the duodenum in a patient with CREST syndrome. However, any etiologic relationship remains to be further investigated.
Functional Identification of NR2 Subunits Contributing to NMDA Receptors on Substance P Receptor-Expressing Dorsal Horn Neurons
Authors: Tong Chi-Kun , Kaftan Edward J , MacDermott Amy B
Published: October 2008

NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers typically composed of two NR1 and two of four NR2 subunits: NR2A-2D. Mice lacking specific NR2 subunits show deficits in pain transmission yet subunit location in the spinal cord remains unclear. We have combined electrophysiological and pharmacological approaches to investigate the composition of functional NMDA receptors expressed by lamina I, substance P receptor-expressing (NK1R+) neurons, as well as NK1R- neurons. Under low Mg2+ conditions (100 μM), the conductance of NMDA receptors at −90 mV (g(−90 mV)) with NR2A or NR2B subunits (NR2A/B) is low compared to conductance measured at the membrane potential where the inward current is maximal or maximal inward current (MIC) (ratio of ~0.07 calculated from Kuner and Schoepfer, 1996). For NR2C or NR2D subunits (NR2C/D), the ratio is higher (ratio ~0.4). NK1R+ and NK1R- neurons express NMDA receptors that give ratios ~0.28 and 0.16, respectively, suggesting both types of subunits are present in both populations of neurons, with NK1R+ neurons expressing a higher percentage of NR2C/D type NMDA receptors. This was confirmed using EAB318, an NR2A/B preferring antagonist, and UBP141, a mildly selective NR2C/D antagonist to increase and decrease the g(−90 mV)/g(MIC) ratios in both subpopulations of neurons.
Competitive Inhibition of Organic Anion Transporting Polypeptide 1c1-Mediated Thyroxine Transport by the Fenamate Class of Nonsteroidal Antiinflammatory Drugs
Authors: Westholm Daniel E. , Stenehjem David D. , Rumbley Jon N. , Drewes Lester R. , Anderson Grant W.
Published: October 2008

Organic anion transporting polypeptide (Oatp) 1c1 is a high-affinity T4 transporter with narrow substrate specificity expressed at the blood-brain barrier. A transport model using cells overexpressing Oatp1c1 was created to identify novel Oatp1c1 substrates and inhibitors. Rat Oatp1c1 was cloned and stably expressed in human embryonic kidney 293 cells. Oatp1c1-transfected human embryonic kidney 293 cells transported 125I-labeled T4 in a time-dependent manner that was completely abolished in the presence of excess unlabeled T4. Next, various compounds, including inhibitors of thyroid hormone uptake, were screened for inhibitory effects on Oatp1c1-mediated T4 uptake. Phenytoin (64%), indocyanine green (17%), fenamic acid (68%), diclofenac (51%), and meclofenamic acid (33%) all reduced T4 uptake by Oatp1c1 when assayed at concentrations of 10 μM. Dose-response assays for the fenamic acids, iopanoic acid, indocyanine green, and phenytoin revealed IC50 values for Oatp1c1 T4 uptake below or near the blood plasma levels after therapeutic doses. Further kinetic assays and reciprocal plot analyses demonstrated that the fenamic acid diclofenac inhibited in a competitive manner. Finally, microvessels were isolated from adult rat brain and assessed for T4 uptake. Ten micromolar of fenamate concentrations inhibited T4 microvessel uptake with a similar hierarchical inhibition profile [fenamic acid (43%), diclofenac (78%), and meclofenamic acid (85%)], as observed for Oatp1c1 transfected cells. Oatp1c1 is expressed luminally and abluminally in the blood-brain barrier endothelial cell, and exhibits bidirectional transport capabilities. Together, these data suggest that Oatp1c1 transports fenamates into, and perhaps across, brain barrier cells.

The fenamate class of nonsteroidal anti-inflammatory drugs is transported by organic anion transporting polypeptides and competitively inhibits thyroxine transport in brain microvessels.
Transcription of Steroidogenic Acute Regulatory Protein in the Rodent Ovary and Placenta: Alternative Modes of Cyclic Adenosine 3′, 5′-Monophosphate Dependent and Independent Regulation
Authors: Yivgi-Ohana Natalie , Sher Noa , Melamed-Book Naomi , Eimerl Sarah , Koler Moriah , Manna Pulak R. , Stocco Douglas M. , Orly Joseph
Published: October 2008

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a −81/−78 sequence, whereas GATA-2 binds to a −66/−61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein β and steroidogenic factor 1 that bind to upstream elements (−117/−95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Proximal cis-elements in the steroidogenic acute regulatory protein (Star) promoter allow versatile transcriptional regulation in tissue-specific manners and differentiation-dependent patterns of STAR expression in rodent ovary and placenta.
Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin
Authors: Pillot Bruno , Soty Maud , Gautier-Stein Amandine , Zitoun Carine , Mithieux Gilles
Published: October 2008

The aim of this study was to assess in rats the effect of protein feeding on the: 1) distribution of endogenous glucose production (EGP) among gluconeogenic organs, and 2) repercussion on the insulin sensitivity of glucose metabolism. We used gene expression analyses, a combination of glucose tracer dilution and arteriovenous balance to quantify specific organ release, and hyperinsulinemic euglycemic clamps to assess EGP and glucose uptake. Protein feeding promoted a dramatic induction of the main regulatory gluconeogenic genes (glucose-6 phosphatase and phosphoenolpyruvate carboxykinase) in the kidney, but not in the liver. As a consequence, the kidney glucose release was markedly increased, compared with rats fed a normal starch diet. Protein feeding ameliorated the suppression of EGP by insulin and the sparing of glycogen storage in the liver but had no effect on glucose uptake. Combined with the previously reported induction of gluconeogenesis in the small intestine, the present work strongly suggests that a redistribution of glucose production among gluconeogenic organs might occur upon protein feeding. This phenomenon is in keeping with the improvement of insulin sensitivity of EGP, most likely involving the hepatic site. These data shed a new light on the improvement of glucose tolerance, previously observed upon increasing the amount of protein in the diet, in type 2 diabetic patients.

Protein feeding increases kidney gluconeogenesis without increasing global endogenous glucose production, and improves insulin suppression of the latter, likely at the hepatic site.
Deletion of the G Protein-Coupled Receptor 30 Impairs Glucose Tolerance, Reduces Bone Growth, Increases Blood Pressure, and Eliminates Estradiol-Stimulated Insulin Release in Female Mice
Authors: Mårtensson Ulrika E. A. , Salehi S. Albert , Windahl Sara , Gomez Maria F. , Swärd Karl , Daszkiewicz-Nilsson Joanna , Wendt Anna , Andersson Niklas , Hellstrand Per , Grände Per-Olof , Owman Christer , Rosen Clifford J. , Adamo Martin L. , Lundquist Ingmar , Rorsman Patrik , Nilsson Bengt-Olof , Ohlsson Claes , Olde Björn , Leeb-Lundberg L. M. Fredrik
Published: October 2008

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30(−/−) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

The G protein-coupled receptor GPR30 maintains normal bone growth, glucose homeostasis, and blood pressure, and mediates estradiol-stimulated insulin release in female mice.
Mutant Parathyroid Hormone-Related Protein, Devoid of the Nuclear Localization Signal, Markedly Inhibits Arterial Smooth Muscle Cell Cycle and Neointima Formation by Coordinate Up-Regulation of p15Ink4b and p27kip1
Authors: Fiaschi-Taesch Nathalie , Sicari Brian , Ubriani Kiran , Cozar-Castellano Irene , Takane Karen K. , Stewart Andrew F.
Published: October 2008

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (ΔNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of ΔNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which ΔNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of ΔNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, ΔNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G0/G1 arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in ΔNLS-PTH-related protein VSMC. More importantly, p15INK4 and p27kip1, two critical inhibitors of the G1/S progression, were markedly increased. Normalization of both p15INK4b and p27kip1 by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15INK4b and p27kip1 fully account for the marked cell cycle slowing induced by ΔNLS-PTH-related protein in VSMCs. Finally, ΔNLS-PTH-related protein is able to induce p15INK4 and p27kip1 expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of ΔNLS-PTH-related protein actions, and suggest that ΔNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.

This study provides the mechanistic underpinnings for understanding how Δ-NLS-PTHrP functions, and suggests that Δ-NLS-PTHrP may have particular efficacy for the prevention of arterial re-stenosis.
A Functional Leptin System Is Essential for Sodium Tungstate Antiobesity Action
Authors: Canals Ignasi , Carmona María C. , Amigó Marta , Barbera Albert , Bortolozzi Analía , Artigas Francesc , Gomis Ramon
Published: October 2008

Sodium tungstate is a novel agent in the treatment of obesity. In diet-induced obese rats, it is able to reduce body weight gain by increasing energy expenditure. This study evaluated the role of leptin, a key regulator of energy homeostasis, in the tungstate antiobesity effect. Leptin receptor-deficient Zucker fa/fa rats and leptin-deficient ob/ob mice were treated with tungstate. In lean animals, tungstate administration reduced body weight gain and food intake and increased energy expenditure. However, in animals with deficiencies in the leptin system, treatment did not modify these parameters. In ob/ob mice in which leptin deficiency was restored through adipose tissue transplantation, treatment restored the tungstate-induced body weight gain and food intake reduction as well as energy expenditure increase. Furthermore, in animals in which tungstate administration increased energy expenditure, changes in the expression of key genes involved in brown adipose tissue thermogenesis were detected. Finally, the gene expression of the hypothalamic neuropeptides, Npy, Agrp, and Cart, involved in the leptin regulation of energy homeostasis, was also modified by tungstate in a leptin-dependent manner. In summary, the results indicate that the effectiveness of tungstate in reducing body weight gain is completely dependent on a functional leptin system.

Anti-obesity activity of tungstate is due to an increase in thermogenesis and a reduction in food intake and depends entirely on a functional leptin system.
N-Type Ca2+-Channels in Murine Pancreatic β-Cells Are Inhibited by an Exclusive Coupling with Somatostatin Receptor Subtype 1
Authors: Smith Paul A.
Published: October 2008

Somatostatin (SRIF) is a well-established inhibitor of insulin secretion, an effect in part mediated by a direct inhibition of voltage-operated Ca2+-channels. However, the identity of the somatostatin receptor subtypes (SSTRs) and voltage-operated Ca2+-channels involved in this process are unknown. Whole-cell perforated patch-clamp methods were applied to the murine pancreatic β-cell line, MIN6, to explore the molecular pharmacology of this problem. SRIF-14 inhibited voltage-gated Ca2+ currents (ICa2+) by 19 ± 3% (n=24) with a pEC50 = 9.05 (95% confidence limits 9–9.1). This action was mimicked solely by 100 nm CH-275, a selective agonist at the somatostatin type 1 receptor (SSTR1), but not by 100 nm BIM-23027, L-362855, or NNC-269100; agonists selective for the other four SSTRs known to exist in MIN6. The inhibition of ICa2+ produced by SRIF and CH-275 was insensitive to pertussis toxin but was reversed by a prepulse to +100 mV. The inhibition of ICa2+ by SRIF-14 was unaffected by 20 μm nifedipine, an inhibitor of L-type Ca2+ channels. Application of the specific N-type Ca2+ channel (Cav2.2) inhibitor ω-conotoxin GV1A at 100 nm mimicked, and as a consequence abolished, the inhibitory effect of SRIF-14 on ICa2+. SRIF selectively inhibits N-type Ca2+-channels in murine pancreatic β-cells via exclusive coupling with SSTR1. These findings help explain how SSTR1 activation can inhibit insulin secretion in pancreatic β-cells and suggest a possible new therapeutic lead for treatment of hyperinsulinemia.

In pancreatic β-cells, somatostatin selectively inhibits N-type, but not other, Ca2+-channels via a direct and exclusive coupling with somatostatin receptor subtype 1.
Expression of Peroxisome Proliferator-Activated Receptor-γ in Key Neuronal Subsets Regulating Glucose Metabolism and Energy Homeostasis
Authors: Sarruf David A. , Yu Fang , Nguyen Hong T. , Williams Diana L. , Printz Richard L. , Niswender Kevin D. , Schwartz Michael W.
Published: October 2008

In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-γ agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARγ is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARγ distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARγ immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARγ immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and α-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARγ deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARγ knockout mice exhibited dramatic reductions of both hypothalamic PPARγ mRNA levels and PPARγ immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARγ mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARγ in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARγ is expressed.

Peroxisome proliferator-activated receptor-γ, a key regulator of adipogenesis and insulin sensitivity in peripheral tissues, is also expressed in neurons involved in body weight control.
Repeated Ferret Odor Exposure Induces Different Temporal Patterns of Same-Stressor Habituation and Novel-Stressor Sensitization in Both Hypothalamic-Pituitary-Adrenal Axis Activity and Forebrain c-fos Expression in the Rat
Authors: Weinberg Marc S. , Bhatt Aadra P. , Girotti Milena , Masini Cher V. , Day Heidi E. W. , Campeau Serge , Spencer Robert L.
Published: October 2008

Repeated exposure to a moderately intense stressor typically produces attenuation of the hypothalamic-pituitary-adrenal (HPA) axis response (habituation) on re-presentation of the same stressor; however, if a novel stressor is presented to the same animals, the HPA axis response may be augmented (sensitization). The extent to which this adaptation is also evident within neural activity patterns is unknown. This study tested whether repeated ferret odor (FO) exposure, a moderately intense psychological stressor for rats, leads to both same-stressor habituation and novel-stressor sensitization of the HPA axis response and neuronal activity as determined by immediate early gene induction (c-fos mRNA). Rats were presented with FO in their home cages for 30 min a day for up to 2 wk and subsequently challenged with FO or restraint. Rats displayed HPA axis activity habituation and widespread habituation of c-fos mRNA expression (in situ hybridization) throughout the brain in as few as three repeated presentations of FO. However, repeated FO exposure led to a more gradual development of sensitized HPA-axis and c-fos mRNA responses to restraint that were not fully evident until after 14 d of prior FO exposure. The sensitized response was evident in many of the same brain regions that displayed habituation, including primary sensory cortices and the prefrontal cortex. The shared spatial expression but distinct temporal development of habituation and sensitization neural response patterns suggests two independent processes with opposing influences across overlapping brain systems.

Repeated exposure of rats to ferret odor leads to rapid development of stimulus-specific habituation and slower development of novel-stressor sensitization of forebrain and hypothalamic-pituitary-adrenal axis activity.
Promotion of β-Cell Differentiation in Pancreatic Precursor Cells by Adult Islet Cells
Authors: Chen Wei , Begum Salma , Opare-Addo Lynn , Garyu Justin , Gibson Thomas F. , Bothwell Alfred L. M. , Papaioannou Virginia E. , Herold Kevan C.
Published: October 2008

It is thought that differentiation of β-cell precursors into mature cells is largely autonomous, but under certain conditions differentiation can be modified by external factors. The factors that modify β-cell differentiation have not been identified. In this study, we tested whether adult islet cells can affect the differentiation process in mouse and human pancreatic anlage cells. We assessed β-cell proliferation and differentiation in mouse and human pancreatic anlage cells cocultured with adult islet cells or βTC3 cells using cellular, molecular, and immunohistochemical methods. Differentiation of murine anlage cells into β-cells was induced by mature islet cells. It was specific for β-cells and not a general feature of endodermal derived cells. β-Cell differentiation required cell-cell contact. The induced cells acquired features of mature β-cells including increased expression of β-cell transcription factors and surface expression of receptor for stromal cell-derived factor 1 and glucose transporter-2 (GLUT-2). They secreted insulin in response to glucose and could correct hyperglycemia in vivo when cotransplanted with vascular cells. Human pancreatic anlage cells responded in a similar manner and showed increased expression of pancreatic duodenal homeobox 1 and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and increased production of proinsulin when cocultured with adult islets. We conclude that mature β-cells can modify the differentiation of precursor cells and suggest a mechanism whereby changes in differentiation of β-cells can be affected by other β-cells.

Mature β cells affect differentiation of pancreatic anlage cells into functional β cells. The differentiated cells respond to glucose and ameliorate diabetes.
Endothelial Dysfunction in Mice with Streptozotocin-induced Type 1 Diabetes Is Opposed by Compensatory Overexpression of Cyclooxygenase-2 in the Vasculature
Authors: Nacci Carmela , Tarquinio Mariela , De Benedictis Leonarda , Mauro Annamaria , Zigrino Addolorata , Carratù Maria Rosaria , Quon Michael J. , Montagnani Monica
Published: October 2008

Cardiovascular complications of diabetes result from endothelial dysfunction secondary to persistent hyperglycemia. We investigated potential compensatory mechanisms in the vasculature that oppose endothelial dysfunction in diabetes. BALB/c mice were treated with streptozotocin (STZ) to induce type 1 diabetes (T1D). In mesenteric vascular beds (MVBs), isolated ex vivo from mice treated with STZ for 1 wk, dose-dependent vasorelaxation to acetylcholine (ACh) or sodium nitroprusside was comparable with that in age-matched control mice (CTRL). By contrast, MVBs from mice treated with STZ for 8 wk had severely impaired vasodilator responses to ACh consistent with endothelial dysfunction. Pretreatment of MVBs from CTRL mice with nitric oxide synthase inhibitor nearly abolished vasodilation to ACh. In MVB from 1-wk STZ-treated mice, vasodilation to ACh was only partially impaired by L-Nω-arginine methyl ester. Thus, vasculature of mice with T1D may have compensatory nitric oxide-independent mechanisms to augment vasodilation to ACh and oppose endothelial dysfunction. Indeed, pretreatment of MVBs isolated from 1-wk STZ-treated mice with NS-398 [selective cyclooxygenase (COX)-2 inhibitor] unmasked endothelial dysfunction not evident in CTRL mice pretreated without or with NS-398. Expression of COX-2 in MVBs, aortic endothelial cells, and aortic vascular smooth muscle cells from STZ-treated mice was significantly increased (vs. CTRL). Moreover, concentrations of the COX-2-dependent vasodilator 6-keto-prostaglandin F-1α was elevated in conditioned media from aorta of STZ-treated mice. We conclude that endothelial dysfunction in a mouse model of T1D is opposed by compensatory up-regulation of COX-2 expression and activity in the vasculature that may be relevant to developing novel therapeutic strategies for diabetes and its cardiovascular complications.

Endothelial dysfunction in type 1 diabetes is opposed by compensatory mechanisms involving increased expression and activity of cyclooxygenase-2 in the vasculature induced by pro-inflammatory signaling.
Lean Phenotype and Resistance to Diet-Induced Obesity in Vitamin D Receptor Knockout Mice Correlates with Induction of Uncoupling Protein-1 in White Adipose Tissue
Authors: Narvaez Carmen J. , Matthews Donald , Broun Emily , Chan Michelle , Welsh JoEllen
Published: October 2008

Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1α-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D3, the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-γ or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D3 and the VDR in the control of adipocyte metabolism and lipid storage in vivo.

Mice lacking the vitamin D receptor or its ligand display reduced adiposity, resistance to diet-induced obesity, and induction of uncoupling protein-1 in white adipose tissue.
The Cyclopentenone 15-Deoxy-Δ12,14-Prostaglandin J2 Delays Lipopolysaccharide-Induced Preterm Delivery and Reduces Mortality in the Newborn Mouse
Authors: Pirianov Grisha , Waddington Simon N. , Lindström Tamsin M. , Terzidou Vasiliki , Mehmet Huseyin , Bennett Phillip R.
Published: October 2008

Intrauterine infection is a common trigger for preterm birth and is also a risk factor for the subsequent development of neurodevelopmental abnormalities in the neonate. Bacterial lipopolysaccharide (LPS) binds to toll-like receptor-4 (TLR-4) to activate proinflammatory signaling pathways, which are implicated in both preterm delivery and antenatal brain injury. The transcription factor nuclear factor-κB (NF-κB) is a key player in the orchestration of the inflammatory response and has a central role in parturition. Here we show that intrauterine administration of TLR-4-specific LPS to pregnant mice results in the activation of NF-κB in the maternal uterus and the fetal brain, up-regulation of proinflammatory proteins cyclooxygenase-2, chemokine ligand 1, ChemoKine (C-C motif) ligand 2, and cytosolic phospholipase A2 in myometrium, and induction of preterm delivery. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an antiinflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We report that coadministration of 15d-PGJ2 and LPS to pregnant mice delays LPS-induced preterm delivery and confers protection from LPS-induced fetal mortality. This is associated with inhibition of myometrial NF-κB, cytosolic phospholipase A2, and c-Jun N-terminal kinase activation, and of inflammatory protein synthesis. Therefore 15d-PGJ2 has anti-inflammatory effects via inhibition of multiple aspects of inflammation-driven TRL-4 signaling pathway. Thus, 15d-PGJ2 or compounds with similar antiinflammatory functions may have potential as therapeutic agents in the management of preterm labor with the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation.

Intrauterine lipopolysaccharide administration to pregnant mice activates nuclear factor-κ and Jun N-terminal kinase, causing preterm labor. 15-deoxy-Δ12,14-prostaglandin J2 inhibits this via multiple aspects of the toll-like receptor-4 signaling pathway.
Commentary: The Year in Basic Science: Update of Estrogen Plus Progestin Therapy for Menopausal Hormone Replacement Implicating Stem Cells in the Increased Breast Cancer Risk
Authors: Horwitz Kathryn B.
Published: October 2008

This transcript is based on my The Year in Basic Science lecture at ENDO 2008. I reviewed current data surrounding hormone replacement therapy and the relationship between systemic estrogen plus progestin (E+P) treatment and increased breast cancer risk, and I explored the hypothesis that women who develop breast cancer while on E+P had occult, undiagnosed disease before they started therapy. Beginning with recent hormone replacement therapy data focusing on E+P and its association with breast cancer to set the stage, the lecture then reviewed our newly published data that progestins expand breast cancer stem cells. Finally, the issues of occult or undiagnosed breast cancer in presumably healthy women, and of tumor dormancy in breast cancer survivors, were brought to bear on the discussion. Taken together, these apparently disparate themes allowed me to suggest the idea that systemic progestins have the ability to reawaken cancers that were presumed to be either nonexistent or cured. To avoid this potentially devastating outcome while retaining the benefits of E+P, I advocated the use of local P delivery methods, rather than the currently popular systemic routes.
Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract
Authors: Chang Ching-Pin , Stankunas Kryn , Shang Ching , Kao Shih-Chu , Twu Karen Y. , Cleary Michael L.
Published: October 2008

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract(OFT). We demonstrate that the homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. We show that Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development.
Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer ofMesp2
Authors: Yasuhiko Yukuto , Kitajima Satoshi , Takahashi Yu , Oginuma Masayuki , Kagiwada Harumi , Kanno Jun , Saga Yumiko
Published: October 2008

The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. Here we report that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. We also generated a knock-in mouse in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespbenhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice.
Ultraviolet Spectroscopy and Theoretical Calculations of Mono- and Dihydrated Clusters of the Guanine Nucleosides: Possibility of Different Hydration Structures for Guanosine and 2′-Deoxyguanosine
Authors: Saigusa Hiroyuki , Mizuno Nobuteru , Asami Hiroya , Takahashi Kenshi , Tachikawa Masanori
Published: October 2008

Mono- and dihydrated clusters of guanosine (Gs) and 2′-deoxyguanosine (2′-dGs) are formed by laser desorption combined with supersonic-jet cooling, and their electronic spectra are obtained by resonant two-photon ionization spectroscopy. The electronic spectra of these hydrated clusters appear to be composed of multiple structural isomers. Possible hydration structures are investigated based on comparison with the result for 9-methylguanine (9MG) and computational studies. The dihydrated cluster of Gs is found to exhibit absorption bands which are significantly shifted with respect to those of 2′-dGs and 9MG. The observation suggests the existence of specific dihydrate structures involving the 2′-hydroxy group of Gs.
Synthesis and Characterization of Ruthenium Complexes Having Tridentate N-Ethyl-N,N-bis(2-pyridylmethyl)amine Coordinating in a Facial or Meridional Fashion
Authors: Shimizu Yasunori , Fukui Sohei , Oi Takao , Nagao Hirotaka
Published: October 2008

Ruthenium complexes containing a tridentate N-ethyl-N,N-bis(2-pyridylmethyl)amine (N,N-bis(2-pyridylmethyl)ethylamine: bpea) ligand having two different types of nitrogen donors, one an amine and the other pyridine rings connected with flexible CH2-arms, were synthesized and characterized. A trichlororuthenium isomeric pair of fac- and mer-[RuCl3(bpea)] was synthesized from RuCl3·nH2O in a H2O–C2H5OH solution. A reaction of fac-[RuCl3(bpea)] in an C2H5OH–H2O–CH3CN solution under refluxing conditions afforded a triacetonitrile complex, fac-[Ru(CH3CN)3(bpea)](PF6)2. Four nitrosylruthenium complexes, trans(NO, py), cis(NO, Cl), fac-[RuCl2(NO)(bpea)]PF6, trans(NO, OH), cis(NO, NO2), mer-[Ru(NO2)(OH)(NO)(bpea)]PF6, trans(NO, OCH3), cis(NO, Cl), mer-[RuCl(OCH3)(NO)(bpea)]PF6 and trans(NO, OH), cis(NO, Cl), mer-[RuCl(OH)(NO)(bpea)]PF6, were synthesized and characterized by X-ray crystallography. The bpea of three nitrosylruthenium complexes bearing an electron-donating ligand such as hydroxo or methoxo as an ancillary ligand coordinated in a meridional fashion.
Preparation, Characterization, and Structures of Dinuclear Cobalt(III) Complexes Triply Bridged by Two μ-Hydroxo and One μ-Carbonato
Authors: Ama Tomoharu , Yonemura Toshiaki , Yoshimura Takahisa
Published: October 2008

Two [Co2(L)2(μ-CO3)(μ-OH)2] complexes (L: N-methyliminodiacetato, or 1,4,7-triazacyclononane) were prepared and characterized. In each complex, the dihedral angle between Co–Oμ-OH–O′μ-OH and Co′–Oμ-OH–O′μ-OH planes was ca. 160° and the μ-CO32− plane was perpendicular to each Co–Oμ-OH–μ-O′μ-OH plane. 13C NMR signals of μ-CO32− in these complexes appeared at ca. 170 ppm.