bluuebunny/crossref_metadata_2025 huggingface dataset:
A cryptic promoter in potato virus X vector interrupted plasmid construction
Authors: Guo Yuyuan , German Thomas L , Schultz Ronald D
Published: March 2007

Background

Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation).

Results

A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR.

Conclusion

It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but inE. coli) can interrupt the downstream work.
Multiple Levels of Control of the Expression of the Human AβH‐J‐J Locus Encoding Aspartyl‐β‐hydroxylase, Junctin, and Junctate
Authors: FERIOTTO GIORDANA , FINOTTI ALESSIA , BREVEGLIERI GIULIA , TREVES SUSAN , ZORZATO FRANCESCO , GAMBARI ROBERTO
Published: March 2007

Abstract:  The human AβH‐J‐J locus is a genomic sequence which generates three functionally distinct proteins, the enzyme aspartyl‐β‐hydroxylase (AβH), the structural protein of sarcoplasmic reticulum junctin, and the membrane‐bound calcium binding protein junctate. The first and second exons are mutually exclusive when mature mRNAs are produced. Moreover, the use of different splice donors has been shown to be involved in the generation of protein diversity by alternative splicing. As to transcriptional regulation, two promoters (P1 and P2) were identified. When the P1 and P2 promoter sequences are compared, important differences are clearly detectable. The most interesting result emerging from studies focused on the P2 promoter is that the calcium‐dependent transcriptional factor MEF‐2 activates the transcription of junctin, junctate, and AβH in excitable tissues and, to a lesser extent, in kidney. No Sp1 binding sites are present in the P2 promoter. In contrast, P1 promoter contains GC‐rich sequences, which have homologies with the Sp1 consensus binding site.
Activation of Nuclear Factor κB by Different Agents
Authors: HELLWEG CHRISTINE E. , ARENZ ANDREA , BOGNER SUSANNE , SCHMITZ CLAUDIA , BAUMSTARK‐KHAN CHRISTA
Published: March 2007

Abstract:  The transcription factor nuclear factor κB (NF‐κB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF‐κB in epithelial cells in response to different stresses, a cell‐based screening assay for activation of NF‐κB‐dependent gene transcription in human embryonic kidney cells (HEK/293) was developed
. This assay allows detection of NF‐κB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell‐based assay, activation of the pathway by several agents, for example, tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF‐κB activation by TNF‐α were examined. NF‐κB was activated by TNF‐α, IL‐1β, PMA, and camptothecin in a dose‐dependent manner, but not by LPS. TNF‐α results in the strongest induction of NF‐κB‐dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20–35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF‐κB activation or direct communication via gap junctions in the cell layer diminishing the TNF‐α response.
The MECP2 Gene Mutation Screening in Rett Syndrome Patients from Croatia
Authors: MATIJEVIĆ TANJA , KNEŽEVIĆ JELENA , BARIŠIĆ INGEBORG , REŠIĆ BISERKA , ČULIĆ VIDA , PAVELIĆ JASMINKA
Published: March 2007

Abstract:  Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder almost exclusively affecting females and is usually sporadic. Mutations in MECP2 gene have been found in more than 80% of females with typical features of RTT. In this study, we analyzed 15 sporadic cases of RTT. In 7 of 15 patients (47%), we detected pathogenic mutations in the coding parts of MECP2 fourth exon. We found two missense (T158M, R133C), two nonsense (R168X, R270X), two frameshift mutations (P217fs and a double deletion of 28‐bp at 1132–1159 and 10‐bp at 1167–1176), and one in‐frame deletion (L383_E392del10). To our knowledge, the last two mutations have not been reported yet. We also detected one previously described polymorphism (S194S). In conclusion, these results show that the fourth exon should be the first one analyzed because it harbors most of the known mutations. Moreover, mutation‐negative cases should be further analyzed for gross rearrangements. This is the first study of its kind in Croatia and it enabled us to give the patients an early confirmation of RTT diagnosis.
Prostaglandins Regulate Transcription by Means of Prostaglandin Response Elements Located in the Promoters of Mammalian Na,K‐ATPase β1 Subunit Genes
Authors: MATLHAGELA KEIKANTSE , TAUB MARY
Published: March 2007

Abstract:  Prostaglandins are potent products of arachidonic acid metabolism that play significant roles in regulating ion transport in the kidney. In the Madin Darby canine kidney (MDCK) cell line prostaglandin E1 (PGE1) stimulates the activity of the Na,K‐ATPase and regulates transcription. Transient transfection studies conducted in MDCK cells with a human Na,K‐ATPase β1 subunit promoter/luciferase construct, pHβ1‐1141 Luc, showed a PGE1 stimulation. The PGE1 stimulation was inhibited by the PGE receptor antagonists SC19220 and AH6809, indicating the involvement of EP1 receptors (coupled to phospholipase C) and EP2 receptors (coupled to adenylate cyclase), respectively. A prostaglandin‐regulatory element (PGRE) within the β1 subunit promoter (−110 to −92, AGTCCCTGC) is sufficient to elicit a PGE1 stimulation in a heterologous promoter (in pLUC‐MCS). Studies with promoter mutants indicated that in addition to the PGRE, an adjacent Sp1 site was also essential for regulation by PGE1. Consistent with the involvement of Sp1 are the results of DNA affinity precipitation studies, which indicate that Sp1 as well as CREB, and Sp3 all bind to the PGRE. The involvement of this PGRE in transcriptional regulation of the Na,K‐ATPase β1 gene was examined in a number of species. Only human and chimpanzee promoters possessed an identical PGRE site, unlike dog, rat, and mouse, which possessed Sp1 sites in similar locations. Two alternative PGREs were subsequently identified. The sequence of the one of these PGREs (TGACCTTC, −445 to −438) was conserved throughout all species examined, suggesting its physiologic significance.
Effects of AT1Receptor‐Mediated Endocytosis of Extracellular Ang II on Activation of Nuclear Factor‐κB in Proximal Tubule Cells
Authors: ZHUO JIA L. , CARRETERO OSCAR A. , LI XIAO C.
Published: March 2007

Abstract: Angiotensin II (Ang II) exerts powerful proinflammatory and growth effects on the development of Ang II‐induced hypertensive glomerulosclerosis and tubulo‐interstitial fibrosis. The proinflammatory and growth actions of Ang II are primarily mediated by activation of cell surface type 1 receptors (AT1) and the transcription factor nuclear factor‐κB (NF‐κB). However, binding of cell surface receptors by extracellular Ang II also induces receptor‐mediated endocytosis of the agonist‐receptor complex in renal cells. The purpose of the present study was to determine whether AT1receptor‐mediated endocytosis of extracellular Ang II is required for Ang II‐induced NF‐κB activation and subsequent proliferation of rabbit renal proximal tubule cells. Expression of AT1(primarily AT1aor human AT1) receptors in these cells was confirmed by Western blot, showing that transfection of a human AT1receptor‐specific 20‐25 nucleotide siRNA knocked down more than 70% of AT1receptor protein (P< 0.01). Stimulation of proximal tubule cells by Ang II (1 nM) induced fourfold increases in NF‐κB activity (P< 0.01). The Ang II‐increased NF‐κB activity was significantly attenuated by coadministration of losartan (10 μM), an AT1receptor‐selective blocker, or colchicine (1 μM), a selective cytoskeleton microtubule inhibitor known to block receptor‐mediated endocytosis (P< 0.01). Furthermore, Ang II significantly increased3H‐thymidine incorporation (>55%,P< 0.01), an index of cell proliferation and DNA synthesis, and the effect was also attenuated by coadministration of losartan and colchicine (P< 0.01). Our results therefore suggest that AT1receptor‐mediated endocytosis of extracellular Ang II may be required for Ang II‐induced NF‐κB activation and subsequent cell proliferation in renal proximal tubule cells.
Effect of Valproic Acid, a Histone Deacetylase Inhibitor, on Cell Death and Molecular Changes Caused by Low‐Dose Irradiation
Authors: ZÁŠKODOVÁ DARINA , ŘEZÁČOVÁ MARTINA , VÁVROVÁ JIŘINA , VOKURKOVÁ DORIS , TICHÝ ALEŠ
Published: March 2007

Abstract:  Valproic acid (VA), a histone deacetylase inhibitor (HDACI), in vitro induces differentiation of promyelocyte leukemia cell (HL‐60) and proliferation arrest and apoptosis of various leukemia cell lines. In MOLT‐4 cells (human T lymphocyte leukemia) the cell cycle arrest is caused by 2 mM VA, while 4 mM VA induces mainly apoptosis. In our work we studied effect of VA on molecular mechanisms responsible for cell cycle arrest (2 mM VA) or apoptosis induction (4 mM VA). The aim of our article was to evaluate a cotreatment by low (cytostatic) concentrations of VA with ionizing radiation and an effect of this combination on apoptosis induction in tumor cells MOLT‐4. We prove that 24‐h long incubation with VA causes acetylation of histones H3 and H4 in concentration‐dependent manners. During first hours after the beginning of cultivation with VA in both studied concentrations (2 and 4 mM) an increase of p53 and its phosphorylation on serine 392 is detected, as well as a phosphorylation of Mdm2 on serine 166. After 8 and 24 h after the beginning of cultivation with 2 mM VA we detect p21, which is not observed after exposure to 4 mM VA. Cleavage of lamin B to 46 kDa fragment as an indicator of apoptosis was apparent after 24‐h long incubation with 4 mM VA. In this article we prove radiosensitizing effect of VA. After 3‐days long cultivation of cells with 2 mM VA the D0 value decreased from 0.7 to 0.2 Gy. Also the EC70 value fell from 0.97 to 0.38 mM when the cells were irradiated with a dose of 1 Gy before the continual cultivation with VA. Continual cultivation of MOLT‐4 cells irradiated by the dose of 1 Gy with VA caused during 14 days after irradiation significant increase of apoptotic cells in comparison to the cells exposed to only one factor. As a conclusion it can be postulated that continual exposure of MOLT‐4 cells to VA increases apoptosis and decreases colony‐forming capacity of the cells irradiated with small dose of radiation.
Arrest of Cancer Cell Proliferation by dsRNAs
Authors: KABILOVA TATYANA O. , VLADIMIROVA AL`BINA V. , CHERNOLOVSKAYA ELENA L. , VLASSOV VALENTIN V.
Published: March 2007

Abstract:  Inhibition of c‐myc and N‐myc genes by dsRNAs in carcinoma and neuroblastoma cells was investigated. siRNA‐Ex3 targeted to the third exon of c‐myc gene was found to decrease the level of c‐myc but not N‐myc mRNA and decrease the rate or even arrest proliferation of c‐myc overexpressing cell lines KB‐3‐1 and SK‐N‐MC. This siRNA did not affect proliferation of IMR‐32 (which overexpress N‐myc). siRNA‐Ex2 corresponding (with 1‐2 mismatches) to the conservative region of the second exon of both c‐ and N‐myc was able to downregulate both genes and to reduce proliferation of KB‐3‐1, SK‐N‐MC, and IMR‐32 cells. Long dsRNA corresponding to the 3 exon of c‐myc gene (dsMyc), poly(I:C), and GU‐rich siRNA‐I, corresponding to the intron sequence of human MDR1 gene demonstrated high antiproliferative activity in experiments with KB‐3‐1 cells. Short‐term elevation of PKR or/and OAS1 mRNA levels was detected in the cells affected by interferon inducer poly(I:C). dsMyc, poly(I:C), and even siRNA‐I, which could not affect c‐myc mRNA by RNA interference mechanism, were found to inhibit proliferation of the KB‐3‐1 cells and to decrease the mRNA level of interferon‐sensitive genes c‐myc and β‐actin.
Design and Functional Activity of Phosphopeptides with Potential Immunomodulating Capacity, Based on the Sequence of Grb2‐Associated Binder 1
Authors: KERTESZ AKOS , TAKACS BALAZS , VARADI GYORGYI , TOTH GABOR K. , SARMAY GABRIELLA
Published: March 2007

Abstract:  A cell membrane permeable phosphopeptide corresponding to the SHP‐2 binding motif of Grb2‐associated binder 1 (Gab1) interferes with the Gab1 adaptor‐dependent functions and modulates B cell receptor‐triggered intracellular signaling in B cell tumors.
Induction of Apoptosis of Osteoclasts by Targeting Transcription Factors with Decoy Molecules
Authors: PIVA ROBERTA , PENOLAZZI LETIZIA , ZENNARO MARGHERITA , BIANCHINI ERCOLINA , MAGRI EROS , BORGATTI MONICA , LAMPRONTI ILARIA , LAMBERTINI ELISABETTA , TAVANTI ELISA , GAMBARI ROBERTO
Published: March 2007

Abstract:  We review the effects of two transcription factor decoy oligonucleotides on apoptosis of human osteoclasts (OCs). The first decoy molecule was designed to inhibit nuclear factor kappa‐B (NF‐κB) binding to target sequence, the second to increase estrogen receptor (ER) α expression. We found that both decoy molecules are potent inducers of apoptosis of human OCs, associated with increase of caspase 3 activity and decrease of interleukin 6 expression. In addition, we provide evidence indicating that these oligonucleotides are active in vivo in inducing OCs apoptosis. Because OCs are essential for skeletal development and remodeling throughout the life of animal and man, the approach described is of potential clinical importance.
Competition Effects Shape the Response Sensitivity and Kinetics of Phosphorylation Cycles in Cell Signaling
Authors: SALAZAR CARLOS , HÖFER THOMAS
Published: March 2007

Abstract:  Phosphorylation cycles are a core component of cell signaling networks. The response sensitivity and kinetics of these cycles are controlled by thermodynamic, kinetic, and structural factors, including binding affinities, catalytic activities, and the phosphorylation order of multiple sites. Based on mathematical models, we interpret the role of these factors in terms of competition effects. For the regulation of a single phosphorylation site, two kinds of competition effects turn out to shape behavior: the competition between kinase and phosphatase to bind the substrate, and the competition between the distinct phosphorylation forms of the substrate for binding to either enzyme. Depending on the concentrations and mutual affinities of the enzymes and the target, the response function can be graded, ultrasensitive, or biphasic. In multiply phosphorylatable proteins, additional factors generating competition effects are present and more complex responses can be obtained. For example, the combination of a cooperative kinetics with the conditions for zero‐order ultrasensitivity may yield a bistable response. We show that a repeated competition between kinase and phosphatase for binding the substrate and/or between the phosphorylation and dephosphorylation reactions at each phosphorylation site generally result in a threshold response. The phosphorylation time is also strongly affected by the kinetic design of the cycle. In particular, threshold responses are generally associated with very long phosphorylation times. We also argue here that a description in terms of elementary binding and reaction steps is required for an appropriate analysis of these cycles in cell signaling.
An Optimal Treatment for Pediatric Graves’ Disease Is Radioiodine
Authors: Rivkees Scott A. , Dinauer Catherine
Published: March 2007

Context: Antithyroid medications, surgery, and radioactive iodine have been used for more than five decades for the treatment of hyperthyroidism due to Graves’ disease in children, adolescents, and adults. Despite the widespread use of these different approaches, controversy still exists relative to the merits of each treatment, especially regarding the use of radioactive iodine.

Objective: The objective of the study was to address the risk and benefits of 131I therapy, as compared with other treatment approaches.

Position: Long-term, spontaneous remission of Graves’ disease occurs in less than 30% of children. Thus, the majority of children with Graves’ disease will need definitive, curative therapy. There is little evidence that use of antithyroid medications beyond 1 or 2 yr increases the likelihood of spontaneous, long-term remission. Although the use of antithyroid medications is standard practice, the use of antithyroid medications involves definite risks. When used at sufficient doses, radioactive iodine is an effective cure for Graves’ disease and is associated with few acute side effects. Potential long-term adverse side effects, including thyroid cancer and genetic damage, have yet to be observed in individuals treated as children or adolescents with 131I.

Conclusion: Properly administered, radioactive iodine remains an ideal form of treatment for Graves’ disease in the pediatric population. Because of the increased risk of thyroid cancer associated with low-dose thyroid irradiation in children, larger, rather than smaller, doses of 131I should be given.
Theoretical Investigation of the Proton Affinity and Gas-Phase Basicity of Neutral x,y-Dihydroxybenzoic Acid and its Derivatives
Authors: Rebber Brandon L. , Halfacre J. Anthony , Beran Kyle A. , Beller Nicholas R. , Gomez Mario , Bashir Sajid , Giannakopulos Anastassios E. , Derrick Peter J.
Published: March 2007

Proton affinities ( PA), gas-phase basicities ( GB) and acidities ( GA), which are some of the important physical properties of a matrix in matrix-assisted laser desorption ionization mass spectrometry, have been calculated using density functional theory (DFT) for a number of dihydroxybenzoic (DHB) acid isomers and derivatives. The theoretical PA and gas-phase basicity ( GB) values for the neutral x,y-DHB acids, ionic radicals, Na+ and K+ salts as well as oxygen- and hydrogen-bridged dimers of x,y-DHB have been calculated. Analysis of the computational data indicates that there are lower PA/ GB values for the anionic dimers compared to the PA/ GB values for the electrically neutral oxygen-bridged dimers. The PA/ GB values for the neutral and radical cations are larger than the neutral monomers and the PA/ GB values for the radical anions are slightly lower than the anionic class of isomers. The PA/ GB values for the salts (x, y-DHB−Na/K+) are significantly higher (100–150 kJ mol−1) than the neutral x,y-DHB acids. The above theoretical results are in agreement with experimental values obtained by Fourier transorm ion cyclotron resonance mass spectrometry employing a thermokinetic method. Correlation of experimentally and theoretically predicted values suggests that this theoretical calculation method could be used to derive information on different matrices.
Quantitative Trait Loci Associated with Photoperiodic Response and Stage of Diapause in the Pitcher-Plant Mosquito,Wyeomyia smithii
Authors: Mathias Derrick , Jacky Lucien , Bradshaw William E , Holzapfel Christina M
Published: March 2007

A wide variety of temperate animals rely on length of day (photoperiodism) to anticipate and prepare for changing seasons by regulating the timing of development, reproduction, dormancy, and migration. Although the molecular basis of circadian rhythms regulating daily activities is well defined, the molecular basis for the photoperiodic regulation of seasonal activities is largely unknown. We use geographic variation in the photoperiodic control of diapause in the pitcher-plant mosquito Wyeomyia smithii to create the first QTL map of photoperiodism in any animal. For critical photoperiod (CPP), we detect QTL that are unique, a QTL that is sex linked, QTL that overlap with QTL for stage of diapause (SOD), and a QTL that interacts epistatically with the circadian rhythm gene, timeless. Results presented here confirm earlier studies concluding that CPP is under directional selection over the climatic gradient of North America and that the evolution of CPP is genetically correlated with SOD. Despite epistasis between timeless and a QTL for CPP, timeless is not located within any detectable QTL, indicating that it plays an ancillary role in the evolution of photoperiodism in W. smithii. Finally, we highlight one region of the genome that includes loci contributing to CPP, SOD, and hormonal regulation of development.
Community Reactions to Campaigns Addressing Crystal Methamphetamine Use among Gay and Bisexual Men in New York City
Authors: Nanín José E. , Parsons Jeffrey T. , Bimbi David S. , Grov Christian , Brown Justin T.
Published: March 2007

Crystal methamphetamine ( aka “crystal meth”) use with high-risk sex has become an emerging health problem for gay and bisexual men in New York City since the late 1990s. Public health campaigns were eventually developed to encourage gay and bisexual men to avoid or reconsider using crystal meth. Reactions to three campaigns were measured with a cross-sectional survey administered in 2004. Among an ethnically-diverse sample of 971 gay and bisexual men, 61.8% reported seeing the campaigns. Those who reported ever using crystal meth, recent use, and recent use with sex were significantly more likely to have seen the campaigns. In general, white men, HIV-negative men, and men not currently using crystal meth responded more positively to the campaigns than their counterparts; yet, more men of color reported having discussions with partners and friends about their crystal use as a result of these campaigns. Implications for researchers and practitioners are discussed.
Boundaries and Dreams
Authors: Hartmann Ernest , Kunzendorf Robert G.
Published: March 2007

This article reviews and summarizes a number of published studies as well as recent unpublished work by the authors on Boundaries (Thin vs. Thick Boundaries) and various aspects of dreaming. Analysis of data by groups as well as by individuals demonstrates a surprisingly high correlation between thinness of boundaries and dream recall frequency. Thin boundaries appears to be the only personality measure clearly related to dream recall frequency. We also demonstrate a relationship between thin boundaries and dream content. Dreams of persons with thin boundaries are rated more dream-like, more emotional, and more bizarre in a number of studies. Thin boundaries are correlated especially with Central Image Intensity—a measure that is elevated in powerful dreams, dreams after traumatic events, and dreams after 9/11/01. These results strengthen our view that the “trait” continuum running from very thick boundaries at one end to very thin boundaries at the other is closely related to the “state” continuum running from focused waking activity at one end, through waking fantasy and daydreaming, to dreaming at the other end.
Epinephrine and insulin stimulate different mitogen-activated protein kinase signaling pathways in rat skeletal muscle.
Authors: Napoli R , Gibson L , Hirshman M F , Boppart M D , Dufresne S D , Horton E S , Goodyear L J
Published: March 2007

Little is known about the regulation of the mitogen-activated protein (MAP) kinase signaling cascades by hormonal stimulation in vivo. The extracellular signal-regulated kinase (ERK) and the c-jun kinase (JNK) are two MAP kinase signaling pathways that could play a role in the cellular response to hormones such as insulin and epinephrine. We studied the effects of insulin (20 U/rat) and epinephrine (25 microg/100 g body wt) injected in vivo on ERK and JNK signaling in skeletal muscle from Sprague-Dawley rats. Insulin significantly increased ERK phosphorylation and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2 (RSK2), by 1.4-fold, but it had no effect on JNK activity. In contrast, epinephrine had no effect on ERK phosphorylation or RSK2 activity, but it increased JNK activity by twofold, an effect that was inhibited by the presence of combined alpha and beta blockade. Furthermore, the phosphorylation of both p46 and p55 isoforms of JNK, measured by phosphospecific antibody, was increased severalfold. The activity and phosphorylation of MAP kinase kinase (MKK)-4, an upstream regulator of JNK, was unchanged by epinephrine. Incubation of isolated soleus muscles in vitro with epinephrine (10(-5) mol/l) also increased JNK activity by twofold. These data are the first to demonstrate that epinephrine can increase JNK activity. Insulin and epinephrine have different effects on MAP kinase signaling pathways in skeletal muscle, which may be one of the underlying molecular mechanisms through which these hormones regulate opposing metabolic functions.
Prevention of diabetes in NOD mice by a mutated I-Ab transgene.
Authors: Singer S M , Tisch R , Yang X D , Sytwu H K , Liblau R , McDevitt H O
Published: March 2007

Susceptibility to the human autoimmune disease IDDM is strongly associated with those haplotypes of the major histocompatibility complex (MHC) carrying DQB1 alleles that do not encode aspartic acid at codon 57. Similarly, in a spontaneous animal model of this disease, the NOD mouse, the genes of the MHC play an important role in the development of diabetes. The DQB1 homolog in NOD mice, I-Ab(g7), encodes a histidine at codon 56 and a serine at codon 57, while all other known I-Ab alleles encode proline and aspartic acid, respectively, at these positions. We therefore mutated the NOD I-Ab allele to encode proline at position 56 and aspartic acid at position 57 and introduced this allele onto the NOD genetic background to study the effect of these substitutions on susceptibility to diabetes. No transgenic mice developed diabetes by 8 months of age, and transgenic mice had markedly reduced lymphocytic infiltration in the pancreas compared with nontransgenic littermates. Furthermore, splenocytes from transgenic mice failed to proliferate or secrete gamma-interferon in response to a panel of beta-cell autoantigens, although the mice did produce beta-cell specific antibodies. Interestingly, the proportion of IgG1 and IgE relative to IgG2a comprising these autoantibodies was much greater in transgenic mice compared with nontransgenic control mice. Finally, T-cells from transgenic mice inhibited the adoptive transfer of diabetes to irradiated recipients. This inhibition was partially reversed by treatment of the recipients with a combination of anti-interleukin (IL)-4 and anti-IL-10 monoclonal antibodies. Thus, a transgenic class II MHC allele encoding aspartic acid at B57 prevents diabetes, in part, by promoting the production of IL-4 and IL-10, which interfere with the effector phase of the diabetic process.
Glycated cholecystokinin-8 has an enhanced satiating activity and is protected against enzymatic degradation.
Authors: O'Harte F P , Mooney M H , Kelly C M , Flatt P R
Published: March 2007

Monoglycated cholecystokinin octapeptide (CCK-8) (glucitol-Asp1 adduct) modified at the NH2-terminus was prepared under hyperglycemic conditions, purified by high-performance liquid chromatography, and characterized by mass spectrometry (Mr 1228.4 Da) and peptide sequencing. CCK-8 (100 nmol/kg, i.p.) significantly (P < 0.001) reduced voluntary food intake of fasted mice for up to 30 min after its administration, compared with saline-administered controls. Glycated CCK-8 reduced food intake at 30-120 min (P < 0.01 to P < 0.001) and significantly reduced feeding compared with CCK-8 from 60 to 120 min (P < 0.01). In vitro plasma degradation studies indicated that glycated CCK-8 was resistant to the normal rapid enzymatic conversion to CCK fragments. This study demonstrated that CCK-8 is a potent short-term inhibitor of food intake, and that structural modification of this peptide by amino-terminal glycation leads to enhanced satiating activity, partially due to increased resistance to serum aminopeptidase degradation.
Plasma glucose levels are reduced in rats and mice treated with an inhibitor of glucose-6-phosphate translocase.
Authors: Parker J C , VanVolkenburg M A , Levy C B , Martin W H , Burk S H , Kwon Y , Giragossian C , Gant T G , Carpino P A , McPherson R K , Vestergaard P , Treadway J L
Published: March 2007

The activity of glucose-6-phosphatase (G-6-Pase) in isolated rat microsomes was inhibited by a new selective inhibitor of the multi-subunit G-6-Pase system, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3,4-dihydroxy-5-(3-imid azo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (compound A) with a 50% inhibitory concentration (IC50) of approximately 10 nmol/l. Compound A (500 nmol/l) inhibited the uptake of [14C]glucose-6-phosphate (G-6-P) into intact isolated rat microsomes, confirming that this agent blocks G-6-P translocation, as suggested by previous studies using intact and permeabilized microsomes. The inhibition of microsomal G-6-P transport by compound A was associated with inhibition of the rate of glucose output from rat hepatocytes incubated in the presence of 25 nmol/l glucagon (IC50 approximately 320 nmol/l.) Compound A (1 micromol/l) also inhibited the basal rate of glucose production by rat hepatocytes by 47%. Intraperitoneal administration of compound A to fasted mice lowered circulating plasma glucose concentrations dose-dependently at doses as low as 1 mg/kg. This effect was comparatively short-lived; glucose lowering was maximal at 30 min after dosing with 100 mg/kg compound A (-71%) and declined thereafter, being reversed within 3 h. A similar time course of glycemic response was observed in fasted rats; glucose lowering was maximal 30 min after dosing with 100 mg/kg compound A (-36%) and declined until the effect was fully reversed by 3 h postdose. In rats subjected to compound A treatment, liver glycogen content was increased. G-6-P and lactate levels were maximally elevated 30 min after dosing and declined thereafter. Cumulatively, these results suggest that the mechanism of glucose lowering by compound A was via inhibition of G-6-Pase activity, mediated through inhibition of the T1 subunit of the microsomal G-6-Pase enzyme system. Drug levels measured over the same time course as that used to assess in vivo efficacy peaked within 30 min of administration, then declined, which is consistent with the transient changes in plasma glucose and liver metabolites.
Normal glucose-induced suppression of glucose production but impaired stimulation of glucose disposal in type 2 diabetes: evidence for a concentration-dependent defect in uptake.
Authors: Nielsen M F , Basu R , Wise S , Caumo A , Cobelli C , Rizza R A
Published: March 2007

The present studies were undertaken to determine whether people with type 2 diabetes are resistant to the effects of glucose as well as insulin. Diabetic and nondiabetic subjects were studied on three occasions. Hormone secretion was inhibited with somatostatin. Insulin concentrations were kept at "basal" levels (referred to as low insulin infusion) from 0 to 180 min then increased to approximately 200 pmol/l from 181 to 360 min (referred to as high insulin infusion). Glucose concentrations were clamped at either approximately 95, approximately 130, or approximately 165 mg/dl on each occasion. In the presence of basal insulin concentrations, a progressive increase in glucose from 95 to 130 to 165 mg/dl was accompanied by a comparable and progressive decrease (P = 0.001 to 0.003 by analysis of variance [ANOVA]) in endogenous glucose production (measured with [6-(3)H]glucose) and total glucose output (measured with [2-(3)H]glucose) and incorporation of 14CO2 into glucose (an index of gluconeogenesis) in both diabetic and nondiabetic subjects, indicating normal hepatic (and perhaps renal) response to glucose. In the nondiabetic subjects, an increase in glucose concentration from 95 to 130 to 165 mg/dl resulted in a progressive increase in glucose disappearance during both the low (19.9 +/- 1.8 to 23.6 +/- 1.8 to 25.4 +/- 1.6 micromol x kg(-1) x min(-1); P = 0.003 by ANOVA) and high (36.4 +/- 3.1 to 47.6 +/- 4.5 to 61.1 +/- 7.0 micromol x kg(-1) x min(-1); P = 0.001 by ANOVA) insulin infusions. In contrast, in the diabetic subjects, whereas an increase in glucose from 95 to 130 mg/dl resulted in an increase in glucose disappearance during both the low (P = 0.001) and high (P = 0.01) dose insulin infusions, a further increase in glucose concentration to 165 mg/dl had no further effect (P = 0.41 and 0.38) on disappearance at either insulin dose (low: 14.2 +/- 0.8 to 18.2 +/- 1.1 to 18.7 +/- 2.4 micromol x kg(-1) x min(-1); high: 21.0 +/- 3.2 to 33.9 +/- 6.4 to 32.5 +/- 8.0 micromol x kg(-1) x min(-1) for 95, 130, and 165 mg/dl, respectively). We conclude that whereas glucose-induced stimulation of its own uptake is abnormal in type 2 diabetes, glucose-induced suppression of endogenous glucose production and output is not. The abnormality in uptake occurs in the presence of both basal and high insulin concentrations and is evident at glucose concentrations above but not below 130 mg/dl, implying a defect in a glucose-responsive step.
Glucose production, utilization, and cycling in response to moderate exercise in obese subjects with type 2 diabetes and mild hyperglycemia.
Authors: Giacca A , Groenewoud Y , Tsui E , McClean P , Zinman B
Published: March 2007

The glucoregulatory and hormonal responses to moderate-intensity exercise (50% VO2max for 45 min) were examined in subjects with type 2 diabetes and mild hyperglycemia. We studied seven obese subjects with type 2 diabetes and seven lean and seven obese control subjects (fasting plasma glucose levels, 7.5 +/- 0.5, 4.8 +/- 0.1, and 5.2 +/- 0.1 mmol/l, respectively). Glucose production, utilization, and cycling (flux between glucose and glucose-6-phosphate [G-6-P]) were measured with [6-(3)H]glucose and [2-(3)H]glucose using the constant specific-activity method. Insulin levels decreased normally during exercise in diabetic subjects. Plasma glucose levels decreased in diabetic subjects, but remained constant in control subjects. Basal glucose production was not different among groups and increased similarly during exercise. The decrease in plasma glucose in diabetic subjects was due to greater glucose utilization (867 +/- 83 vs. 726 +/- 143 micromol x m(-2) x min(-1); P < 0.05). This was a consequence of the mass effect of hyperglycemia, since glucose metabolic clearance increased similarly in all groups. Glucose cycling, expressed as a percentage of total glucose output (i.e., flux through G-6-P) was elevated at rest (P < 0.01), but decreased during exercise (P < 0.01). The catecholamine response to exercise was blunted in diabetic subjects, presumably indicating autonomic dysfunction. In conclusion, during moderate-intensity exercise in obese diabetic subjects with mild hyperglycemia, 1) insulin secretory responses were normally regulated; 2) glucose homeostasis was different from that in nondiabetic subjects because glucose levels decreased during exercise; 3) the decrease in plasma glucose was due to greater-than-normal rates of glucose utilization, which were sustained by hyperglycemia; and 4) elevated basal rates of glucose cycling decreased during exercise, presumably because exercise simultaneously lowered plasma glucose, was associated with a blunted catecholamine response, and accentuated an underlying defect in hepatic glucokinase activity in type 2 diabetes.
A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and PPAR-gamma: effect of PPAR-alpha activation on abnormal lipid metabolism in liver of Zucker fatty rats.
Authors: Murakami K , Tobe K , Ide T , Mochizuki T , Ohashi M , Akanuma Y , Yazaki Y , Kadowaki T
Published: March 2007

We investigated the biological activity of a novel thiazolidinedione (TZD) derivative, KRP-297, and the molecular basis of this activity. When administered to obese Zucker fatty rats (obese rats) at 10 mg/kg for 2 weeks, KRP-297, unlike BRL-49,653, restored reduced lipid oxidation, that is, CO2 and ketone body production from [14C]palmitic acid, in the liver by 39% (P < 0.05) and 57% (P < 0.01), respectively. KRP-297 was also significantly more effective than BRL-49,653 in the inhibition of enhanced lipogenesis and triglyceride accumulation in the liver. To understand the molecular basis of the biological effects of KRP-297, we examined the effect on peroxisome proliferator-activated receptor (PPAR) isoforms, which may play key roles in lipid metabolism. Unlike classical TZD derivatives, KRP-297 activated both PPAR-alpha and PPAR-gamma, with median effective concentrations of 1.0 and 0.8 micromol/l, respectively. Moreover, radiolabeled [3H]KRP-297 bound directly to PPAR-alpha and PPAR-gamma with dissociation constants of 228 and 326 nmol/l, respectively. Concomitantly, KRP-297, but not BRL-49,653, increased the mRNA and the activity (1.5-fold [P < 0.01] and 1.8-fold [P < 0.05], respectively) of acyl-CoA oxidase, which has been reported to be regulated by PPAR-alpha, in the liver. By contrast, KRP-297 (P < 0.05) was less potent than BRL-49,653 (P < 0.01) in inducing the PPAR-gamma-regulated aP2 gene mRNA expression in the adipose tissues. These results suggest that PPAR-alpha agonism has a protective effect against abnormal lipid metabolism in liver of obese rats.
Alterations in non-insulin-mediated glucose uptake in the elderly patient with diabetes.
Authors: Forbes A , Elliott T , Tildesley H , Finegood D , Meneilly G S
Published: March 2007

It is increasingly recognized that alterations in non-insulin-mediated glucose uptake (NIMGU) play an important pathogenic role in disorders of carbohydrate metabolism. This study was conducted to determine whether NIMGU is impaired in elderly patients with type 2 diabetes. Healthy elderly control subjects (n = 19, age 76 +/- 1 years, BMI 26.8 +/- 1.1 kg/m2) and elderly patients with type 2 diabetes (n = 19, age 76 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2) underwent a 240-min glucose clamp study. Octreotide was infused to suppress endogenous insulin release, and tritiated glucose methodology was used to measure glucose uptake and disposal rates. For the first 180 min, glucose was kept at fasting levels. From 180 to 240 min, glucose was increased to 11 mmol/l. At fasting glucose levels, glucose uptake was similar in both groups. However, glucose clearance was reduced in patients with diabetes (control 1.68 +/- 0.05 ml x kg(-1) x min(-1); diabetes 1.34 +/- 0.07 ml x kg(-1) x min(-1), P < 0.0001). During hyperglycemia, glucose uptake was reduced in patients with diabetes (control 3.16 +/- 0.09 mg x kg(-1) x min(-1); diabetes 2.57 +/- 0.11 mg x kg(-1) x min(-1), P < 0.0001). Peripheral glucose effectiveness (SG) was less in patients with diabetes (control 1.28 +/- 0.04 ml x kg(-1) x min(-1); diabetes 0.94 +/- 0.08 ml x kg(-1) x min(-1), P < 0.0001). Hepatic glucose output and hepatic SG were not different between groups. We conclude that the effect of glucose on glucose uptake is impaired in elderly patients with type 2 diabetes, a finding that may have therapeutic implications for this patient population.
Impact of nocturnal hypoglycemia on hypoglycemic cognitive dysfunction in type 1 diabetes.
Authors: Fanelli C G , Paramore D S , Hershey T , Terkamp C , Ovalle F , Craft S , Cryer P E
Published: March 2007

To test the hypothesis that glycemic thresholds for cognitive dysfunction during hypoglycemia, like those for autonomic and symptomatic responses, shift to lower plasma glucose concentrations after recent antecedent hypoglycemia in patients with type 1 diabetes mellitus (T1DM), 15 patients were studied on two occasions. Cognitive functions were assessed during morning hyperinsulinemic stepped hypoglycemic clamps (85, 75, 65, 55, and 45 mg/dl steps) after, in random sequence, nocturnal (2330-0300) hypoglycemia (48 +/- 2 mg/dl) on one occasion and nocturnal euglycemia (109 +/- 1 mg/dl) on the other. Compared with nondiabetic control subjects (n = 12), patients with T1DM had absent glucagon (P = 0.0009) and reduced epinephrine (P = 0.0010), norepinephrine (P = 0.0001), and neurogenic symptom (P = 0.0480) responses to hypoglycemia; the epinephrine (P = 0.0460) and neurogenic symptom (P = 0.0480) responses were reduced further after nocturnal hypoglycemia. After nocturnal hypoglycemia, in contrast to nocturnal euglycemia, there was less deterioration of cognitive function overall (P = 0.0065) during hypoglycemia based on analysis of the sum of standardized scores (z-scores). There was relative preservation of measures of pattern recognition and memory (the delayed non-match to sample task, P = 0.0371) and of attention (the Stroop arrow-word task, P = 0.0395), but not of measures of information processing (the paced serial addition task) or declarative memory (the delayed paragraph recall task), after nocturnal hypoglycemia. Thus, glycemic thresholds for hypoglycemic cognitive dysfunction, like those for autonomic and symptomatic responses to hypoglycemia, shift to lower plasma glucose concentrations after recent antecedent hypoglycemia in patients with T1DM.
Expression of mRNAs encoding uncoupling proteins in human skeletal muscle: effects of obesity and diabetes.
Authors: Bao S , Kennedy A , Wojciechowski B , Wallace P , Ganaway E , Garvey W T
Published: March 2007

To explore the potential role of the uncoupling protein (UCP) family in human obesity and diabetes, we have used the reverse transcription-polymerase chain reaction to quantify UCP mRNA expression in human skeletal muscle. Levels of mRNA for UCP2, and for both short (UCP3S) and long (UCP3L) forms of UCP3, were highly correlated in individuals, indicating that gene transcription of these UCPs may be coordinately regulated by common mechanisms. In normal glucose-tolerant individuals, muscle UCP2 mRNA levels were positively correlated with percentage of body fat and with BMI (r = 0.6 and P < 0.05 for both). UCP3S mRNA levels were also positively correlated with percentage of body fat (r = 0.52, P < 0.05), and UCP3L mRNA tended to increase as a function of obesity (0.05 < P < 0.1). UCP mRNA levels, however, were not correlated with resting metabolic rate. UCP3S and UCP3L mRNA levels (P < 0.05) and the UCP2 mRNA level (P = 0.09) were increased by 1.8- to 2.7-fold in type 2 diabetes, an effect that could not be explained by obesity. No significant difference was found for UCP2, UCP3S, or UCP3L mRNA levels between insulin-sensitive and insulin-resistant nondiabetic subgroups. We conclude that 1) skeletal muscle mRNA levels encoding UCP2 and UCP3 are correlated among individuals and may be coordinately regulated; 2) UCP3 expression is not regulated by differential effects on UCP3L and UCP3S forms of the mRNA; and 3) UCP mRNA expression tends to increase in muscle as a function of obesity but not of resting metabolic rate or insulin resistance, and is increased in patients with type 2 diabetes.
Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation.
Authors: Magun R , Gagnon A , Yaraghi Z , Sorisky A
Published: March 2007

We have used the 3T3-L1 and 3T3-F442A preadipocyte cell lines to examine the expression and regulation of neuronal apoptosis inhibitory protein (NAIP) during adipocyte differentiation. When 3T3-L1 preadipocytes differentiated into adipocytes, they developed resistance to apoptosis induced by growth factor deprivation, as assessed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling. Protein expression of NAIP was markedly elevated in 3T3-L1 and 3T3-F442A adipocytes compared with that in their fibroblast-like precursors. NAIP was also present in rat white adipocytes. In 3T3-L1 cells, the increase in NAIP occurred by day 4 of the 8-day differentiation protocol, which includes exposure of confluent preadipocytes to insulin, dexamethasone, and isobutylmethylxanthine. Incubation of confluent 3T3-L1 preadipocytes with any of these components alone had no effect on NAIP expression. When 3T3-C2 cells, a control cell line that does not differentiate, were subjected to the differentiation protocol, the low NAIP levels remained unaltered. Addition of rapamycin, a p70 S6 kinase inhibitor that blocks adipocyte differentiation, to the 3T3-L1 differentiation medium prevented the rise in NAIP expression. These data demonstrate for the first time that NAIP is expressed in adipocyte cell lines and primary adipocytes. The differentiation-dependent augmentation of NAIP protein levels in 3T3-L1 adipocytes is closely correlated with the development of resistance to apoptosis induced by growth factor deprivation, suggesting a potential role for NAIP in these cells.
Palmitate and myristate selectively mimic the effect of glucose in augmenting insulin release in the absence of extracellular Ca2+.
Authors: Komatsu M , Sharp G W
Published: March 2007

Under Ca2+-free conditions, activation of the pancreatic beta-cell with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) is permissive for the augmentation of insulin release by glucose and other nutrients. The ability of fatty acids to mimic the effect of glucose and thereby augment insulin secretion in the absence of extracellular Ca2+ is the focus of the present study. In the absence of extracellular Ca2+, glucose, palmitate, and myristate had no effect on insulin release. When, under Ca2+-free conditions, the islets were treated with forskolin to raise cyclic AMP levels and activate protein kinase A and with TPA to activate protein kinase C, glucose, palmitate, and myristate all augmented release to approximately the same extent. No other saturated fatty acid with chain lengths in the C = 6-22 range augmented the release of insulin. This selective augmentation by palmitate or myristate was not seen with forskolin alone, and was seen slightly with TPA and strongly with the combination of forskolin and TPA. The response, which developed slowly and had a time course similar to that of second-phase insulin release, was abolished by the physiological inhibitor norepinephrine. The results suggest that the mechanism underlying the Ca2+-independent augmentation of insulin release by glucose and other nutrients involves the proposed malonyl-CoA/long-chain acyl-CoA pathway with specificity for myristoyl- and palmitoyl-CoA esters and/or their derivatives.