bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of recombination in quasispecies populations of a Hepatitis C Virus patient undergoing anti-viral therapy
Authors: Moreno María P , Casane Didier , López Lilia , Cristina Juan
Published: October 2006

Background/Aim

Hepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. HCV circulates in vivo as a complex population of different but closely related viral variants, commonly referred to as a quasispecies. The extent to which recombination plays a role in the evolution of HCV quasispecies when patients are undergoing anti-viral therapy is currently unknown. In order to gain insight into these matters, we have performed a phylogenetic analysis of HCV quasispecies populations from six patients undergoing anti-viral therapy.

Methods

Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative recombinant sequence as a query. Statistical support for the presence of a recombination event was done by the use of LARD program.

Results

A crossing-over event in the NS5A gene in a HCV strain recovered after four weeks of treatment was identified in quasispecies from a patient with sustained response. Putative parental-like strains were identified as strains circulating in previous weeks on the same patient.

Conclusion

Only one recombinant strain was detected in all patient quasispecies populations studied. The recombination break-point is situated on the PKR-binding region of NS5A. Although recombination may not appeared to be extensive in NS5A genes of HCV quasispecies populations of patients undergoing antiviral therapy, this possibility should be taken into account as a mechanism of genetic variation for HCV.
Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors
Authors: Moriyama Etsuko N , Strope Pooja K , Opiyo Stephen O , Chen Zhongying , Jones Alan M
Published: October 2006

To identify divergent seven-transmembrane receptor (7TMR) candidates from the Arabidopsis thaliana genome, multiple protein classification methods were combined, including both alignment-based and alignment-free classifiers. This resolved problems in optimally training individual classifiers using limited and divergent samples, and increased stringency for candidate proteins. We identified 394 proteins as 7TMR candidates and highlighted 54 with corresponding expression patterns for further investigation.
Inhibition of c-Jun N-terminal kinase rescues influenza epitope-specific human cytolytic T lymphocytes from activation-induced cell death
Authors: Mehrotra Shikhar , Chhabra Arvind , Hegde Upendra , Chakraborty Nitya G , Mukherji Bijay
Published: October 2006

Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen-specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope-specific CTL are quite sensitive to activation-induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen-specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a “self” epitope might be more prone to AICD for maintaining self-tolerance, viral antigen-specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein-derived MP58–66 epitope-specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone or by soluble Ig-Fc chimeras of the death receptors [Fas, TNF receptor (TNF-R), TRAIL-RI, TRAIL-RII]. However, the MP58–66-specific CTL can be rescued from AICD by the c-jun-N-terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope-specific CTL from AICD.
The inflammatory cytokine response of cholesterol-enriched macrophages is dampened by stimulated pinocytosis
Authors: Li Yankun , Tabas Ira
Published: October 2006

Two features of advanced atherosclerotic lesions are large numbers of macrophages and a heightened state of inflammation. Some of the macrophages appear to be enriched with free cholesterol (FCMφs), and we have shown that this process induces the synthesis and secretion of inflammatory cytokines, including TNF-α and IL-6. However, lesions contain many other macrophages that are not FC-enriched (non-FCMφs). Therefore, we sought to understand how the interaction of these two populations of macrophages would influence the inflammatory response. We show here that non-FCMφs possess a robust ability to deplete TNF-α and IL-6 secreted by FCMφs. The mechanism involves enhanced pinocytic uptake and lysosomal degradation of the FCMφ-secreted cytokines by the non-FCMφs. The FCMφs contribute directly to this process by secreting pinocytosis-stimulatory factors that act on non-FCMφs but not on the FCMφs themselves. One of these pinocytosis-stimulatory factors is M-CSF, which is induced by a process involving cholesterol trafficking to the endoplasmic reticulum and signaling through PI-3K and ERK MAPK pathways. However, one or more other FCMφ-secreted factors are also required for stimulating pinocytosis in non-FCMφs. Thus, FCMφs secrete inflammatory cytokines as well as factors that promote the eventual pinocytosis and degradation of these cytokines by neighboring macrophages. This process may normally serve to prevent prolonged or disseminated effects of inflammatory cytokines during inflammation. Moreover, possible perturbation of stimulated pinocytosis during the progression of advanced atherosclerosis may contribute to the heightened inflammatory state of these lesions.
Differential involvement of NF-κB and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils
Authors: Cloutier Alexandre , Ear Thornin , Blais-Charron Emilie , Dubois Claire M , McDonald Patrick P
Published: October 2006

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-κB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-κB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-κB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-κB and selected MAPK pathways. Our data also raise the possibility that NF-κB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.
Sex Hormone Status May Modulate Rate of Expansion of Proximal Femur Diameter in Older Women alongside Other Skeletal Regulators
Authors: Kaptoge S. , Dalzell N. , Folkerd E. , Doody D. , Khaw K.-T. , Beck T. J. , Loveridge N. , Mawer E. B. , Berry J. L. , Shearer M. J. , Dowsett M. , Reeve J.
Published: October 2006

Context: Little is known of associations between hip geometry and skeletal regulators. This is important because geometry is a determinant of both hip function and resistance to fracture.

Objective: We aimed to determine the effects of sex hormone status and other candidate regulators on hip geometry and strength.

Subjects and Methods: A random sample of 351 women aged 67–79 had two to four hip dual-energy x-ray absorptiometry scans performed over 8 yr of follow-up. Hip structural analysis software was used to measure subperiosteal diameter (PD) and the distance from the center of mass to the lateral cortical margin (d-lat) on three 5-mm-thick cross-sectional regions: narrow neck, intertrochanter, and shaft. Section modulus (Z), bone mineral density (grams per centimeter squared), and an index of bone mineral content (cross-sectional area) were calculated as estimators of bone strength. Serum analytes measured at baseline included SHBG, estradiol, PTH, creatinine, albumin, vitamin D metabolites, and glutamate- and γ-carboxyglutamate-osteocalcin (OC). A linear mixed model was used to model associations with predictor variables, including testing whether the predictors significantly modified the effect of aging.

Results: Aging was associated with increasing PD and d-lat, and higher baseline SHBG significantly modified this effect, in the case of PD, increasing the rates of change at the narrow neck region by 19% for SHBG level 2 sd higher than population mean (P = 0.026). Higher baseline creatinine was independently associated with faster increases in PD and d-lat with aging (P < 0.041). Z declined faster with aging if baseline PTH was higher, and higher albumin had a contrary effect. Z was positively associated with free estradiol and inversely associated with SHBG and glutamate-OC.

Conclusion: These results show large effects of SHBG on the regulation of proximal femur expansion and bending resistance, probably acting as a surrogate for low bioavailable estrogen. Potentially important effects for fracture resistance in old age were also revealed for PTH, markers related to renal function and the nutritional markers albumin and undercarboxylated OC.
Prevalence of Autoantibody-Negative Diabetes Is Not Rare at All Ages and Increases with Older Age and Obesity
Authors: Wang Jian , Miao Dongmei , Babu Sunanda , Yu Jeesuk , Barker Jennifer , Klingensmith Georgeanna , Rewers Marian , Eisenbarth George S. , Yu Liping
Published: October 2006

Objective: A significant percentage of nonautoimmune forms of diabetes presents among children in all age groups, with a remarkable increase with age.

Design: From October 1992 to October 2004, a total of 859 children less than 18 yr of age were newly diagnosed with diabetes at the Barbara Davis Center for Childhood Diabetes and had blood samples obtained within 2 wk of disease onset for analysis of antiislet autoantibodies to glutamic acid decarboxylase-65, insulinoma-associated antigen-2, insulin, and islet cell autoantibodies. The relationship of autoantibody positivity with human leukocyte antigen (HLA) class II, body mass index (BMI), glycosylated hemoglobin, age, and ethnicity was analyzed.

Results: Overall 19% (159 of 859) of these children with newly diagnosed diabetes were negative for all autoantibodies, and autoantibody negativity was significantly increased with age (P < 0.01). The Hispanic and Black subjects had significantly increased autoantibody negativity among older children with higher BMI than White subjects. The patients with the highest risk HLA genotype, DR3-DQ2/DR4-DQ8, were significantly less autoantibody negative (P = 0.001), whereas the HLA-protective allele, DQB1*0602, was significantly increased among the autoantibody-negative patients (P < 0.0001). Insulin autoantibodies were dramatically age dependent and were inversely correlated with age in both prevalence (P < 0.0001) and levels (P < 0.0001). Autoantibody positivity was inversely correlated with both BMI and age using multivariate analysis (P < 0.0001 and P = 0.0078, respectively).

Conclusions: A significant percentage of children newly diagnosed with diabetes are negative for all antiislet autoantibodies with a marked increase in obesity-associated autoantibody-negative diabetes after age 10, suggesting diabetes heterogeneity at all ages.
High-Fat/Low-Carbohydrate Diet Reduces Insulin-Stimulated Carbohydrate Oxidation but Stimulates Nonoxidative Glucose Disposal in Humans: An Important Role for Skeletal Muscle Pyruvate Dehydrogenase Kinase 4
Authors: Chokkalingam K. , Jewell K. , Norton L. , Littlewood J. , van Loon L. J. C. , Mansell P. , Macdonald I. A. , Tsintzas K.
Published: October 2006

Aim: The aim of this report was to study the effect of high-fat (HF)/low-carbohydrate (CHO) diet on regulation of substrate metabolism in humans.

Methods: Ten healthy men consumed either a HF (75% energy as fat) or control (35%) diet for 6 d in random order. On d 7, blood glucose disappearance rate (Rd) was determined before and during a hyperinsulinemic euglycemic clamp. Substrate oxidation was determined by indirect calorimetry. Muscle biopsies were obtained prediet, postdiet, and postclamps.

Results: Rd was similar under basal conditions but slightly elevated (∼10%, P < 0.05) during the last 30 min of the clamp after the HF diet. HF diet reduced CHO oxidation under basal (by ∼40%, P < 0.05) and clamp conditions (by ∼20%, P < 0.05), increased insulin-mediated whole-body nonoxidative glucose disposal (by 30%, P < 0.05) and muscle glycogen storage (by ∼25%, P < 0.05). Muscle pyruvate dehydrogenase complex activity was blunted under basal and clamp conditions after HF compared with control (P < 0.05) and was accompanied by an approximately 2-fold increase (P < 0.05) in pyruvate dehydrogenase kinase 4 (PDK4) mRNA and protein expression.

Conclusion: Short-term HF/low-CHO dietary intake did not induce whole-body insulin resistance, but caused a shift in im glucose metabolism from oxidation to glycogen storage. Insulin-stimulated CHO oxidation and muscle pyruvate dehydrogenase complex activity were blunted after the HF diet. Up-regulation of muscle PDK4 expression was an early molecular adaptation to these changes, and we showed for the first time in healthy humans, unlike insulin-resistant individuals, that insulin can suppress PDK4 but not PDK2 gene expression in skeletal muscle.
Differential Gene Expression Profile in Omental Adipose Tissue in Women with Polycystic Ovary Syndrome
Authors: Cortón Marta , Botella-Carretero José I. , Benguría Alberto , Villuendas Gemma , Zaballos Angel , San Millán José L. , Escobar-Morreale Héctor F. , Peral Belén
Published: October 2006

Context: The polycystic ovary syndrome (PCOS) is frequently associated with visceral obesity, suggesting that omental adipose tissue might play an important role in the pathogenesis of the syndrome.

Objective: The objective was to study the expression profiles of omental fat biopsy samples obtained from morbidly obese women with or without PCOS at the time of bariatric surgery.

Design: This was a case-control study.

Settings: We conducted the study in an academic hospital.

Patients: Eight PCOS patients and seven nonhyperandrogenic women submitted to bariatric surgery because of morbid obesity.

Interventions: Biopsy samples of omental fat were obtained during bariatric surgery.

Main Outcome Measure: The main outcome measure was high-density oligonucleotide arrays.

Results: After statistical analysis, we identified changes in the expression patterns of 63 genes between PCOS and control samples. Gene classification was assessed through data mining of Gene Ontology annotations and cluster analysis of dysregulated genes between both groups. These methods highlighted abnormal expression of genes encoding certain components of several biological pathways related to insulin signaling and Wnt signaling, oxidative stress, inflammation, immune function, and lipid metabolism, as well as other genes previously related to PCOS or to the metabolic syndrome.

Conclusion: The differences in the gene expression profiles in visceral adipose tissue of PCOS patients compared with nonhyperandrogenic women involve multiple genes related to several biological pathways, suggesting that the involvement of abdominal obesity in the pathogenesis of PCOS is more ample than previously thought and is not restricted to the induction of insulin resistance.
Tyrosine phosphorylation of p145met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells
Authors: Yamamoto Natsumi , Mammadova Gunay , Song Robert X.-D. , Fukami Yasuo , Sato Ken-ichi
Published: October 2006

Here we address the molecular mechanism of serum-independent survival and growth of human bladder carcinoma cell line 5637. Serum starvation promoted tyrosine phosphorylation of a 145-kDa protein and activation of the tyrosine kinase Src and the receptor for epidermal growth factor (EGFR) over a slow time course (>8 hours). The phosphorylated 145-kDa protein was identified as the β-subunit of c-Met/hepatocyte growth factor (HGF) receptor, p145met, in which tyrosine residues 1003, 1234, and 1235 were phosphorylated. Inhibitors of Src (PP2, SU6656) or EGFR (AG99), but not p145met (K252a), effectively blocked tyrosine phosphorylation of p145met and promoted cell death accompanied by activation of caspase-like proteases. Conditioned medium from the serum-starved 5637 cells or purified EGF readily promoted the activation of Src and EGFR, and tyrosine phosphorylation of p145met in normally grown 5637 cells, suggesting that autocrine signaling of EGFR ligands is responsible for signal transduction events in serum-starved cells. Consistent with this idea, a monoclonal antibody against EGFR that would interfere with the ligand binding to EGFR blocked tyrosine phosphorylation events and promoted the caspase activation and cell death in serum-free conditions. Such apoptotic cell death was also induced by pretreatment of cells with a high concentration of HGF that downregulated endogenous p145met. Nevertheless, Cu2+ ions, competitive inhibitors for HGF-binding to p145met, did not show any effect on cellular functions in serum-free conditions. These results suggest that the serum-independent growth of 5637 cells involves the transmembrane signaling cascade via EGFR ligand(s) (but not HGF), EGFR, Src and p145met.
Synthesis of Copper Phthalocyanine-containing Hyperbranched Polymer Starting from 1,2-Bis(3,4-dicyanophenoxy)benzene and CuCl
Authors: Kwon Young , Hayakawa Teruaki , Kakimoto Masa-aki
Published: October 2006

A novel soluble hyperbranched polymer with copper phthalocyanine (CuPc) was successfully prepared by copper fusion technique of 1,2-bis(3,4-dicyanophenoxy)benzene and CuCl. The structure of copper phthalocyanine-containing hyperbranched polymer confirmed by IR, UV–vis absorption spectra. This polymer showed good solubility in DMF, DMAc, and high thermal stability.
Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells
Authors: Calabrese J. Mauro , Sharp Phillip A.
Published: October 2006

Studies of mammalian RNA interference (RNAi) have focused largely on the actions of microRNAs; however, in other organisms, endogenous short-interfering RNAs (siRNAs) are involved in silencing processes. To date, similar molecules have been difficult to characterize in mammalian cells. P19 is a plant suppressor of RNA silencing that binds with high affinity to siRNAs. Here, the short RNAs bound by P19 in mouse embryonic stem (ES) cells have been characterized. We show that P19 selectively immunoprecipitates endogenous short RNAs from ES cells. Cloning of immunoprecipitated RNA reveals a strong selection for short RNAs that are exact matches to ribosomal RNA (rRNA), with particular short rRNA species highly enriched in P19 immunoprecipitates. Complementary strands to the enriched rRNAs were not cloned, which was surprising because P19 was previously thought to bind only siRNAs. We show that P19 binds tightly to a noncanonical dsRNA substrate comprised of a short RNA annealed to a much longer partner, such that the double-stranded region between the two is 19 base pairs long. Binding to similar endogenous species might explain the association of P19 with short rRNAs in ES cells. Finally, we show that the P19-enriched rRNAs are not involved in canonical RNAi, as they exist in the absence of Dicer and do not function as post-transcriptional gene silencers. Our results support the previous observation that endogenous siRNAs are not abundant molecules in mouse ES cells.
New patterns in genetic and congenital otonephropathies
Authors: Bergstrom Lavonne , Thompson Patricia , Wood Raymond P.
Published: October 2006

In a series of chronic renal and congenitally deaf patients 24 were identified as having inborn renal and otologic disease. Sixteen patients, representing 14 families, had genetic disorders. Only two had the features of Alport's syndrome.

The patients were classified as follows: 1) Probable Alport's — 2 patients (1 family); 2) Atypical hereditary nephritis and sensorineural hearing loss — 7 patients; 3) Renal and inner ear anomalies — 1 patient; 4) Renal, inner ear and multiple anomalies — 4 patients. The temporal bone pathology in one case showed primary neural atrophy and a mild Mondini malformation. In another a Scheibe defect and unusual calcific structures were found in the cochlear duct. 5) Renal, external or middle ear and multiple anomalies — 6 patients (5 families); 6) Renal, middle and inner ear anomalies and multiple anomalies — 2 patients. A temporal bone obtained from one case showed combined middle and inner ear defects. In the other, who had a chromosome defect, predominantly middle ear anomalies were found. 7) Nephrotic syndrome and congenital hearing loss — 1 patient; 8) Unclassified — 1 patient.

Some cases represent entities apparently not previously described. Probably most interesting is the delineation of hereditary nephritis and deafness distinct from Alport's disease.
Bleomycin: Its utilization in the treatment of head and neck cancer
Authors: Bone Robert C. , Byfield John E. , Feren Alan P. , Seagren Stephen L.
Published: October 2006

Originally isolated as an antibiotic, bleomycin is a complex mixture of glycopeptides currently utilized against a variety of malignancies, among them epidermoid cancer of the head and neck. Because of unique independent pharmacological properties, bleomycin may be effectively used at several points in the natural history of tumors. Use of bleomycin as a single agent, as an adjunct to surgery and irradiation and as a palliative drug in head and neck cancer, is now advocated in scattered reports. The rationale for the utilization of bleomycin at the University of California, San Diego and San Diego Veterans Administration Hospitals is discussed. In addtion, examples from the authors' personal series of both response and adverse effects, suggestions for appropriate clinical monitoring to detect early toxicity, and tennets for management of side effects are included in the presentation.
Treatment of spastic dysphonia by recurrent laryngeal nerve section
Authors: Barton Richard Thomas
Published: October 2006

The problem of management of patients with spastic dysphonia has been complicated by a general resistance to speech therapy, psychotherapy, hypnotherapy, and drug therapy. Dedo introduced the concept of recurrent laryngeal nerve section in an attempt to eliminate the hyperfunction and excessive adduction of the vocal folds.

Eleven patients were treated by RLN section with satisfactory results in 8 and some improvement in the other 3. The operation was found to be generally uncomplicated and required on average 4 days of hospitalization.

Dedo's theory that spastic dysphonia is caused by a neurotropic viral‐induced proprioceptive nerve deficit represents a new search for organic cause. His most recent report of finding unmyelinated fibres in one‐third of the resected recurrent laryngeal nerves is of questionable significance. The evidence of deep emotional conflict and/or compulsive lifestyle is found in the majority of the patients, but the syndrome is not typical of an hysterical or conversion neurosis.

Regardless of etiologic theory, RLN section is an effective treatment in selected, longstanding, and resistant instances of spastic dysphonia.
The effect of static tympanic pressure gradients on hearing sensitivity in normal subjects
Authors: Truswell William H. , Randolph Kenneth J. , Snyder G. Gordon
Published: October 2006

The effects of static tympanic air pressure gradients on hearing sensitivity was studied by introducing overpressures of 100 mm H2O to 400 mm H2O into the ear canals of 22 normal subjects. Sweep frequency threshold measures were obtained using a pressure‐tight probe transducer driven by a Bekesy automatic audiometer. These gradients of tympanic pressure disequilibrium produced threshold losses through 4000 Hz, most prominently for the region of 500 to 1000 Hz, that increased systematically with the degree of overpressure. The data may be useful in estimating the degree of conductive loss associated with middle ears presenting retraction without effusion and normally compliant though negatively skewed tympanograms.
The revised learning process questionnaire: A validation of a Western model of students' study approaches to the South Pacific context using confirmatory factor analysis
Authors: Phan Huy P. , Deo Bisun.
Published: October 2006

Background. Research evidence seems to suggest that the social and cultural environments influence students' approaches to their study. This social and cultural contention has led to the rethinking and reconceptualization of theories (e.g. Biggs, 1987; Marton & Säljö, 1976) pertaining to student approaches to learning (SAL) in academic settings.

Aims. The present research discusses two separate empirical studies on student learning approaches situated in the South Pacific region with two respective cohorts of secondary students. Study I involved the examination of secondary Pacific Islands students in their learning approaches using a modified version of Biggs' (1987) original Learning Process Questionnaire (LPQ). Study II involved the administration of a revised version of the LPQ (R‐LPQ‐2F; Kember, Biggs, & Leung, 2004) to another cohort of secondary Pacific Islands students.

Sample. The first sample included 2,150 (1,285 girls, 865 boys) students and the second sample included 2,295 (1,363 girls, 932 boys) students.

Methods. The factor structures of approaches to learning were examined by means of confirmatory factor analysis (CFA) using the LISREL program. Different a priori models were hypothesized and tested.

Results. Results of Study I indicated a two‐factor structure solution to Biggs' LPQ, supporting Richardson's (1994) theoretical model of learning and emphasized the factors of Reproducing and Meaning. Study II indicated a hierarchical organization of two main study approaches – deep and surface – that are structured as higher‐order factors and a defined by four first‐order factors.

Conclusion. The results from the two studies accentuate the important argument for the rethinking and reconceptualization of learning approaches, as well as for the redevelopment and modification of learning inventories such as the LPQ. They also suggest the importance of situating the theoretical paradigm of learning approaches in a social and cultural environment.
Isoform-Specific Early Trafficking of AMPA Receptor Flip and Flop Variants
Authors: Coleman Sarah K. , Möykkynen Tommi , Cai Chunlin , von Ossowski Lotta , Kuismanen Esa , Korpi Esa R. , Keinänen Kari
Published: October 2006

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-Dflopreceptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-Dflipundergoes complex glycosylation and reaches the plasma membrane at >10× higher levels than GluR-Dflop, as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-Dflopnear to the level exhibited by GluR-Dflip. Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.
Auditory Brainstem Timing Predicts Cerebral Asymmetry for Speech
Authors: Abrams Daniel A. , Nicol Trent , Zecker Steven G. , Kraus Nina
Published: October 2006

The left hemisphere of the human cerebral cortex is dominant for processing rapid acoustic stimuli, including speech, and this specialized activity is preceded by processing in the auditory brainstem. It is not known to what extent the integrity of brainstem encoding of speech impacts patterns of asymmetry at cortex. Here, we demonstrate that the precision of temporal encoding of speech in auditory brainstem predicts cerebral asymmetry for speech sounds measured in a group of children spanning a range of language skills. Results provide strong evidence that timing deficits measured at the auditory brainstem negatively impact rapid acoustic processing by specialized structures of cortex, and demonstrate a delicate relationship between cortical activation patterns and the temporal integrity of cortical input.
DNA Polymerase-β Is Expressed Early in Neurons of Alzheimer's Disease Brain and Is Loaded into DNA Replication Forks in Neurons Challenged with β-Amyloid
Authors: Copani Agata , Hoozemans Jeroen J. M. , Caraci Filippo , Calafiore Marco , Van Haastert Elise S. , Veerhuis Robert , Rozemuller Annemieke J. M. , Aronica Eleonora , Sortino Maria Angela , Nicoletti Ferdinando
Published: October 2006

Cultured neurons exposed to synthetic β-amyloid (Aβ) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-β (DNA pol-β) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Aβ-treated neurons, we demonstrate that DNA pol-β coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-β is loaded into neuronal DNA replication forks after Aβ treatment. In response to Aβ the canonical DNA-synthesizing enzyme DNA pol-δ also was loaded into neuronal replication forks, but at later times than DNA pol-β. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-β during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-β and Cdc45, indicating that DNA pol-β loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Aβ, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death.

These data demonstrate that DNA pol-β is an essential component of the DNA replication machinery in Aβ-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-β, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-β in the pathogenesis of AD.
Estrogen Upregulates T-Type Calcium Channels in the Hypothalamus and Pituitary
Authors: Qiu Jian , Bosch Martha A. , Jamali Khalid , Xue Changhui , Kelly Martin J. , Rønnekleiv Oline K.
Published: October 2006

Low voltage-activated (T-type) Ca2+channels are responsible for generating low-threshold spikes (LTS) that facilitate burst firing and transmitter release in neurons. The T-type Ca2+channels contain a regulatory α1 subunit, and several isoforms of the α1 subunit (Cav3.1, 3.2, 3.3) have been cloned. The Cav 3.1 α1 subunit is abundantly expressed in the hypothalamus. Previously, we found that 17 β-estradiol (E2) increased the number of arcuate neurons expressing LTS. Therefore, we used an ovariectomized female guinea pig model to measure the distribution and regulation of Cav3.1 mRNA expression by E2. Guinea pig Cav3.1 α1 subunit sequences, which were cloned by PCR, were used in ribonuclease protection (RPA) andin situhybridization assays to evaluate mRNA expression. Based on a RPA, E2 significantly increased the mRNA expression of Cav3.1 α1 subunit in the mediobasal hypothalamus and the pituitary.In situhybridization analysis revealed that E2 significantly increased Cav 3.1 mRNA expression in medial preoptic nuclei, bed nuclei stria terminalis, and the arcuate nucleus. Whole-cell patch recordings in arcuate neurons revealed that E2 treatment significantly increased the peak T-type Ca2+current density by twofold without affecting the activation/inactivation characteristics and augmented the rebound excitation by threefold to fourfold. These results suggest that estrogen regulates the mRNA expression of T-type calcium channels, which leads to increased functional expression of the channel. Increased expression of T-type channels could be one mechanism by which estrogen augments burst firing and transmitter release in hypothalamic neurons.
Specificity of M and L Cone Inputs to Receptive Fields in the Parvocellular Pathway: Random Wiring with Functional Bias
Authors: Buzás Péter , Blessing Esther M. , Szmajda Brett A. , Martin Paul R.
Published: October 2006

Many of the parvocellular pathway (PC) cells in primates show red–green spectral selectivity (cone opponency), but PC ganglion cells in the retina show no anatomical signs of cone selectivity. Here we asked whether responses of PC cells are compatible with “random wiring” of cone inputs. We measured long-wavelength-sensitive (L) and medium-wavelength-sensitive (M) cone inputs to PC receptive fields in the dorsal lateral geniculate of marmosets, using discrete stimuli (apertures and annuli) to achieve functional segregation of center and surround. Receptive fields between the fovea and 30° eccentricity were measured.

We show that, in opponent PC cells, the center is dominated by one (L or M) cone type, with normally <20% contribution from the other cone type (high “cone purity”), whereas non-opponent cells have mixed L and M cone inputs to the receptive field center. Furthermore, opponent response strength depends on the overall segregation of L and M cone inputs to center and surround rather than exclusive input from one cone type to either region. These data are consistent with random wiring. The majority of PC cells in both foveal (<8°) and peripheral retina nevertheless show opponent responses. This arises because cone purity in the receptive field surround is at least as high as in the center, and the surround in nearly all opponent PC cells is dominated by the opposite cone type to that which dominates the center. These functional biases increase the proportion of opponent PC cells, but their anatomical basis is unclear.
Olfactory Ensheathing Cells Do Not Exhibit Unique Migratory or Axonal Growth-Promoting Properties after Spinal Cord Injury
Authors: Lu Paul , Yang Hong , Culbertson Maya , Graham Lori , Roskams A. Jane , Tuszynski Mark H.
Published: October 2006

Olfactory ensheathing cells (OECs) have been reported to migrate long distances and to bridge lesion sites, guiding axonal regeneration after spinal cord injury (SCI). To understand mechanisms of OEC migration and axonal guidance, we injected lamina propria OECs 1 mm rostral and caudal to C4 SCI sites. One month later, OECs formed an apparent migrating cell tract continuously extending from the injection site through the lesion, physically bridging the lesion. Confocal immunolabeling demonstrated that, whereas this cell tract displaced host astrocytes, descending or ascending long tract axons did not preferentially extend into the cell tract and OECs failed to support bridging of corticospinal axons. Notably, the “bridging” tract of OECs formed within 1 h of cell injection, raising the possibility that cells passively spread from the pressure injection site rather than actively migrating. Control injections of bone marrow stromal cells (MSCs) or fibroblasts 1 mm from the lesion site also rapidly dispersed into the lesion cavity. Cell tracts extending into the lesion site were not seen when cells were injected either at low volumes, into spinal cord gray matter, or 3 d before or 9 d after SCI. OECs proliferated in injection sites, cell tracts, and lesion sites, indicating that OECs can also accumulate through cell proliferation. Thus, OECs do not appear to exhibit significant migratory properties when grafted to the spinal cord, exhibit no detectable difference in promoting axon growth into a SCI site compared with MSCs or fibroblasts, and do not support bridging of corticospinal axons beyond a dorsal column lesion.
Integration of Auditory and Visual Communication Information in the Primate Ventrolateral Prefrontal Cortex
Authors: Sugihara Tadashi , Diltz Mark D. , Averbeck Bruno B. , Romanski Lizabeth M.
Published: October 2006

The integration of auditory and visual stimuli is crucial for recognizing objects, communicating effectively, and navigating through our complex world. Although the frontal lobes are involved in memory, communication, and language, there has been no evidence that the integration of communication information occurs at the single-cell level in the frontal lobes. Here, we show that neurons in the macaque ventrolateral prefrontal cortex (VLPFC) integrate audiovisual communication stimuli. The multisensory interactions included both enhancement and suppression of a predominantly auditory or a predominantly visual response, although multisensory suppression was the more common mode of response. The multisensory neurons were distributed across the VLPFC and within previously identified unimodal auditory and visual regions (O'Scalaidhe et al., 1997; Romanski and Goldman-Rakic, 2002). Thus, our study demonstrates, for the first time, that single prefrontal neurons integrate communication information from the auditory and visual domains, suggesting that these neurons are an important node in the cortical network responsible for communication.
Messergebnisse und Simulationen der Lichtreflexion an Tropfen bei der Messung mit Fasersensoren (Results of Measurement and Simulation of the Light Reflection from Drops on Fibre Sensors)
Authors: Ji Aiming , Wiggers Helmut , Walzel Peter
Published: October 2006

Diese Arbeit stellt eine Messtechnik vor zur Charakterisierung von Mehrphasenströmungen mit einem optischen Fasersensor. Gemessen wird der Verlauf des Tropfenaufpralls auf dem Sensorende. Der komplexe Signalverlauf wird anhand zweier Simulationen erklärt. Die experimentellen Ergebnisse zeigen eine gute Übereinstimmung mit den Berechnungen. Ein Modell der Totalreflexion am Wasserrest, der auf dem Faserende verbleibt, erklärt eine bestimmte Signaleigenschaft.
RET/PTC activation in papillary thyroid carcinoma: European Journal of Endocrinology Prize Lecture
Authors: Santoro Massimo , Melillo Rosa Marina , Fusco Alfredo
Published: October 2006

Papillary thyroid carcinoma (PTC) is frequently associated with RET gene rearrangements that generate the so-called RET/PTC oncogenes. In this review, we examine the data about the mechanisms of thyroid cell transformation, activation of downstream signal transduction pathways and modulation of gene expression induced by RET/PTC. These findings have advanced our understanding of the processes underlying PTC formation and provide the basis for novel therapeutic approaches to this disease.
Characterization and expression of different pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptors in rat ovarian follicles
Authors: Vaccari Sergio , Latini Stefania , Barberi Marzia , Teti Anna , Stefanini Mario , Canipari Rita
Published: October 2006

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide transiently expressed in preovulatory follicles. PACAP acts by interacting with three types of PACAP receptors. PACAP type I receptor (PAC1-R), which binds specifically to both PACAPs and vasoactive intestinal polypeptide (VIP), although with lower affinity, and two VIP receptors, VPAC1-R and VPAC2-R, which bind to PACAP and VIP with equal affinity. In the present study, we showed the expression of all three receptors in whole ovaries obtained from juvenile and gonadotropin-treated immature rats. A more detailed analysis on cells from preovulatory follicles showed that PAC1-R and VPAC2-R were expressed in granulosa cells, whereas only VIP receptors were expressed in theca/interstitial (TI) cells and fully grown oocytes presented only PAC1-R. The distribution of the VIP receptors was confirmed by immunofluorescence. HCG treatment induced stimulation of PAC1-R in granulosa cells and VPAC2-R in TI cells. The presence of functional PACAP/VIP receptors was also supported by metabolic studies. We further evaluated the presence of PACAP and VIP receptors by testing the effect of these peptides on apoptosis in granulosa cells cultured, isolated or in whole follicles. Treatment of follicles with PACAP and VIP dose-dependently inhibited apoptosis, while only PACAP significantly inhibited isolated granulosa cells. These results demonstrate a different expression of PACAP/VIP receptors in the various follicle compartments and suggest a possible role for PACAP and VIP on granulosa and TI cells, both during follicle development and ovulation.
Adenosine does not bind to the growth hormone secretagogue receptor type-1a (GHS-R1a)
Authors: Carreira Marcos C , Camiña Jesus P , Díaz-Rodríguez Esther , Alvear-Perez Rodrigo , Llorens-Cortes Catherine , Casanueva Felipe F
Published: October 2006

Ghrelin regulates GH secretion and energy homeostasis through the GH secretagogue receptor type-1a (GHS-R1a). This G-protein coupled receptor shows the peculiarity to transduce information provided not just by ghrelin as well as by adenosine through a supposed binding site different from the characterized ghrelin-binding pocket. Indeed, adenosine triggers intracellular calcium rise through a distinct signaling pathway to the one described for ghrelin, although it fails to stimulate GH secretion. Despite multiple active conformations of GHS-R1a, suggested as an explanation for a ligand-dependent activation of the downstream signaling, the concept of adenosine as agonist for GHS-R1a has been re-evaluated. The results revealed that calcium rise of both ghrelin and adenosine appears to be mediated by receptors that did not show the same sensitivity to protein kinase C (PKC) activity in GHS-R1a-transfected HEK 293 cells (HEK-GHS-R1a cells). The binding analyses showed the same number of adenosine-binding sites in both HEK 293 (Bmax = 2.01 ± 0.15 fmol/cell) and HEK-GHS-R1a cells (Bmax = 1.90 ± 0.11 fmol/cell). This binding was unaltered by different GHS-R1a antagonists. Western blot analysis showed a similar endogenous expression of endogenous adenosine receptor type-2b and -3 in both cell lines. The Kd values for adenosine were 1.78 μM in HEK 293 cells and 6.30 μM in HEK-GHS-R1a cells, pointing to a modification of agonist affinity induced by overexpression of the GHS-R1a. Additionally, adenosine failed to induce the GHS-R1a endocytosis, although it attenuates the ghrelin-induced GHS-R1a endocytosis. In conclusion, adenosine is not an agonist of the GHS-R1a and its action is mediated by the endogenous adenosine receptor type-2b and -3, which is able to partially use the intracellular signaling machinery of HEK-GHS-R1a cells.
Pup removal suppresses estrogen-induced surges of LH secretion and activation of GnRH neurons in lactating rats
Authors: Fukushima Atsushi , Yin Ping , Ishida Maho , Sugiyama Nobuhiro , Arita Jun
Published: October 2006

During lactation, the suckling stimulus exerts profound influences on neuroendocrine regulation in nursing rats. We examined the acute effect of pup removal on the estrogen-induced surge of LH secretion in ovariectomized lactating rats. Lactating and nonlactating cyclic female rats were given an estradiol-containing capsule after ovariectomy, and blood samples were collected through an indwelling catheter for serum LH determinations. In lactating, freely suckled ovariectomized rats, estrogen treatment induced an afternoon LH surge with a magnitude and timing comparable to those seen in nonlactating rats. Removal of pups from the lactating rats at 0900, 1100, or 1300 h, but not at 1500 h, suppressed the estrogen-induced surge that normally occurs in the afternoon of the same day. The suppressive effect of pup removal at 0900 h was completely abolished when the pups were returned by 1400 h. In contrast, pup removal was ineffective in abolishing the stimulatory effect of progesterone on LH surges. Double immunohistochemical staining for gonadotropin-releasing hormone (GnRH) and c-Fos, a marker for neuronal activation, revealed a decrease, concomitantly with the suppression of LH surges, in the number of c-Fos-immunoreactive GnRH neurons in the preoptic regions of nonsuckled rats. An LH surge was restored in nonsuckled rats when 0.1 μg oxytocin was injected into the third ventricle three times at 1-h intervals during pup removal. These results suggest that the GnRH surge generator of lactating rats requires the suckling stimulus that is not involved in nonlactating cyclic female rats.