bluuebunny/crossref_metadata_2025 huggingface dataset:
Repetitive Transcranial Magnetic Stimulation for Tinnitus: A Case Study
Authors: Richter Gresham T. , Mennemeier Mark , Bartel Twyla , Chelette Kenneth C. , Kimbrell Timothy , Triggs William , Dornhoffer John L.
Published: October 2006

Objectives/Hypothesis: Correlate subjective improvements in tinnitus severity with restoration of cortical symmetry and sustained attention after neuronavigated low‐frequency, repetitive transcranial magnetic stimulation (rTMS).

Study Design: Case study.

Methods: Positron emission tomography and computed tomography imaging (PET‐CT) guided rTMS was performed on a 43‐year‐old white male with more than a 30 year history of bilateral tinnitus. rTMS was administered to the area of increased cortical activation visualized on PET‐CT at a rate of 1 Hz for 30 minutes (1,800 pulses/session) for each of 5 consecutive days, with optimization applied on day 5 using single pulses of TMS to temporarily alter tinnitus perception. Subjective tinnitus severity was rated before and after rTMS using the tinnitus severity index with analogue scale. Attention and vigilance were assessed before and after therapy using the psychomotor vigilance task (PVT), a simple reaction time test that is sensitive to thalamocortical contributions to sustained attention. Posttherapy PET‐CT was used to evaluate any change in asymmetric cortical activation.

Results: The most marked reduction in tinnitus severity occurred after rTMS optimization; this persisted up to 4 weeks after rTMS. PVT testing showed the patient exhibited a statistically significant improvement in mean slowest 10% reaction times after rTMS (P = .004). PET‐CT imaging 2 days after the cessation of rTMS showed no changes in cortical blood flow or metabolic asymmetries.

Conclusions: Low‐frequency rTMS applied to the primary auditory cortex can reduce tinnitus severity, with rTMS optimization yielding the most favorable results. Beneficial changes occurring in the patient's slowest reaction times suggest that attentional deficits associated with tinnitus may also respond to low‐frequency rTMS.
Control of cellular senescence by CPEB
Authors: Groisman Irina , Ivshina Maria , Marin Veronica , Kennedy Norman J. , Davis Roger J. , Richter Joel D.
Published: October 2006

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16INK4A; however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.
Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication
Authors: Lazzerini Denchi Eros , Celli Giulia , de Lange Titia
Published: October 2006

We report that mouse liver cells are highly resistant to extensive telomere dysfunction. In proliferating cells, telomere dysfunction results in chromosome end fusions, a DNA damage signal, and apoptosis or senescence. To determine the consequences of telomere dysfunction in noncycling cells, we used conditional deletion of the telomeric protein TRF2 in hepatocytes. TRF2 loss resulted in telomeric accumulation of γ-H2AX and frequent telomere fusions, indicating telomere deprotection. However, there was no induction of p53 or apoptosis, and liver function appeared unaffected. Furthermore, the loss of TRF2 did not compromise liver regeneration after partial hepatectomy. Remarkably, liver regeneration occurred without cell division involving endoreduplication and cell growth, thereby circumventing the chromosome segregation problems associated with telomere fusions. We conclude that nondividing hepatocytes can maintain and regenerate liver function despite substantial loss of telomere integrity.
The Vibrio harveyi quorum-sensing system uses shared regulatory components to discriminate between multiple autoinducers
Authors: Waters Christopher M. , Bassler Bonnie L.
Published: October 2006

The quorum-sensing bacterium Vibrio harveyi produces and responds to three autoinducers (AIs), and this sensory information converges to control the expression of bioluminescence, biofilm formation, type III secretion (TTS), and protease production. The AIs are detected by cognate sensor histidine kinases that all relay phosphate to the shared response regulator LuxO. LuxO indirectly represses the master regulator of quorum sensing, LuxR, through the activation of multiple genes encoding small regulatory RNAs (called qrr genes for Quorum Regulatory RNA). Here we use differential fluorescence induction to identify 50 quorum-sensing-controlled promoters. Some promoters only showed significant responses in the simultaneous presence of all three AIs, while others displayed substantial responses to the individual AIs. A differential response to each AI input state was also observed for qrr and luxR expression and LuxR protein production. Individual cell analyses revealed that, in each case, all the bacteria in the population respond in unison to the various AI inputs. We propose that the V. harveyi quorum-sensing transition is not switch-like but rather operates in a graded manner, and that this signaling arrangement, which uses shared regulatory proteins, nonetheless provides V. harveyi a mechanism to respond uniquely to different AI input states.
Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo
Authors: Evsikov Alexei V. , Graber Joel H. , Brockman J. Michael , Hampl Aleš , Holbrook Andrea E. , Singh Priyam , Eppig John J. , Solter Davor , Knowles Barbara B.
Published: October 2006

Fully grown oocytes (FGOs) contain all the necessary transcripts to activate molecular pathways underlying the oocyte-to-embryo transition (OET). To elucidate this critical period of development, an extensive survey of the FGO transcriptome was performed by analyzing 19,000 expressed sequence tags of the Mus musculus FGO cDNA library. Expression of 5400 genes and transposable elements is reported. For a majority of genes expressed in mouse FGOs, homologs transcribed in eggs of Xenopus laevis or Ciona intestinalis were found, pinpointing evolutionary conservation of most regulatory cascades underlying the OET in chordates. A large proportion of identified genes belongs to several gene families with oocyte-restricted expression, a likely result of lineage-specific genomic duplications. Gene loss by mutation and expression in female germline of retrotransposed genes specific to M. musculus is documented. These findings indicate rapid diversification of genes involved in female reproduction. Comparison of the FGO and two-cell embryo transcriptomes demarcated the processes important for oogenesis from those involved in OET and identified novel motifs in maternal mRNAs associated with transcript stability. Discovery of oocyte-specific eukaryotic translation initiation factor 4E distinguishes a novel system of translational regulation. These results implicate conserved pathways underlying transition from oogenesis to initiation of development and illustrate how genes acquire and lose reproductive functions during evolution, a potential mechanism for reproductive isolation.
The many faces of amnesia
Authors: Gold Paul E.
Published: October 2006

Results from studies of retrograde amnesia provide much of the evidence for theories of memory consolidation. Retrograde amnesia gradients are often interpreted as revealing the time needed for the formation of long-term memories. The rapid forgetting observed after many amnestic treatments, including protein synthesis inhibitors, and the parallel decay seen in long-term potentiation experiments are presumed to reveal the duration of short-term memory processing. However, there is clear and consistent evidence that the time courses obtained in these amnesia experiments are highly variable within and across experiments and treatments. The evidence is inconsistent with identification of basic temporal properties of memory consolidation. Alternative views include modulation of memory and emphasize the roles that hormones and neurotransmitters have in regulating memory formation. Of related interest, converging lines of evidence suggest that inhibitors of protein synthesis and of other biochemical processes act on modulators of memory formation rather than on mechanisms of memory formation. Based on these findings, memory consolidation and reconsolidation studies might better be identified as memory modulation and “remodulation” studies. Beyond a missing and perhaps unattainable time constant of memory consolidation, some current views of memory consolidation assume that memories, once formed, are generally unmodifiable. It is this perspective that appears to have led to the recent interest in memory reconsolidation. But the view adopted here is that memories are continually malleable, being updated by new experiences and, at the same time, altering the memories of later experiences. Studies of memory remodulation offer promise of understanding the neurobiological bases by which new memories are altered by prior experiences and by which old memories are altered by new experiences.
Amnesia or retrieval deficit? Implications of a molecular approach to the question of reconsolidation
Authors: Miller Courtney A. , Sweatt J. David
Published: October 2006

Post-retrieval interference with a memory has uncovered a phenomenon known to the field as reconsolidation. In this article, we will review the specific molecular mechanisms that have been implicated in reconsolidation. As a result of numerous studies over the past five years, it can now be said with a fair amount of certainty that reconsolidation is not a recapitulation of the mechanisms underlying consolidation, despite what the term “reconsolidation” may suggest. Therefore, in addition to reviewing the known mechanisms of reconsolidation, we will propose that two experimental approaches involving the targeting of specific molecular mechanisms, and the study of these mechanisms during retrieval, may serve useful to the field as it is now able to advance beyond comparisons between consolidation and reconsolidation.
Different parameters support generalization and discrimination learning in Drosophila at the flight simulator
Authors: Brembs Björn , Hempel de Ibarra Natalie
Published: October 2006

We have used a genetically tractable model system, the fruit fly Drosophila melanogaster to study the interdependence between sensory processing and associative processing on learning performance. We investigated the influence of variations in the physical and predictive properties of color stimuli in several different operant-conditioning procedures on the subsequent learning performance. These procedures included context and stimulus generalization as well as color, compound, and conditional discrimination (colors and patterns). A surprisingly complex dependence of the learning performance on the colors’ physical and predictive properties emerged, which was clarified by taking into account the fly-subjective perception of the color stimuli. Based on estimates of the stimuli’s color and brightness values, we propose that the different tasks are supported by different parameters of the color stimuli; generalization occurs only if the chromaticity is sufficiently similar, whereas discrimination learning relies on brightness differences.
Dynamic shifts in corticostriatal expression patterns of the immediate early genes Homer 1a and Zif268 during early and late phases of instrumental training
Authors: Hernandez Pepe J. , Schiltz Craig A. , Kelley Ann E.
Published: October 2006

Adaptive motor actions require prior knowledge of instrumental contingencies. With practice, these actions can become highly automatic in nature. However, the molecular and anatomical substrates mediating these related forms of learning are not understood. In the present study, we used in situ hybridization to measure the mRNA levels of two immediate early genes (IEGs) in an instrumental paradigm where rats learned to lever-press for food. We report that after three training sessions, Homer 1a and Zif268 (an effector and regulatory IEG, respectively) were significantly induced within an extensive corticostriatal network relative to untrained controls. With extended training (23 sessions), however, a shift in the expression patterns of the two genes was evident. Expression of Homer 1a (official symbol Homer1) decreased significantly in frontal and cingulate cortices, whereas striatal expression was generally maintained. Interestingly, Homer 1a expression markedly increased with extensive training in the ventrolateral region of the striatum (VLS) relative to early learners, suggesting that plasticity in the VLS is required for the efficient production of the learned behavior or in habit formation. Zif268 (official symbol Egr1) expression generally decreased with extensive training; however, these changes were not significant. These results demonstrate for the first time, on a molecular level, a dynamic shift in the contribution of corticostriatal systems mediating the early acquisition and consolidation of goal-directed responses to those engaged after extensive training.
Staining Membrane-Bound Proteins with Colloidal Gold
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy
Published: October 2006

INTRODUCTION

Colloidal Gold is the most sensitive staining technique for proteins bound on membranes, detecting as little as 1-3 ng of protein. Protein spots are permanently stained a dark red after incubation with the Colloidal Gold solution. Colloidal Gold staining can detect proteins on both nitrocellulose and PVDF membranes, but it is not recommended for nylon membranes.
Purification of Peptides from Solid-Phase Peptide Synthesis with RP-HPLC
Authors: Boysen Reinhard I. , Hearn Milton T.W.
Published: October 2006

INTRODUCTION

Purification of peptides derived from solid-phase peptide synthesis (SPPS) requires the removal of deletion peptides resulting from incomplete coupling/deprotection steps, from racemization or side-chain rearrangement, and from various chemical substances introduced during the deprotection or cleavage stages of an SPPS procedure. This protocol details the purification and analysis of many synthetic peptides of 2-65 amino acid residues. These peptides contain a number of ionizable or polar side chains, but do not contain secondary structural elements (such as β-sheets) that favor supramolecular assembly.
Fragmentation of Protein Using Trypsin
Authors: Simpson Richard J.
Published: October 2006

INTRODUCTION

In this protocol, the highly specific protease trypsin is used to hydrolyze a protein completely. Proteolysis is carried out with high levels of trypsin to ensure total proteolysis. (Trypsin is a robust enzyme; hence, proteolysis can also be carried out under denaturing conditions such as 2 M guanidine-HCl, 0.1% SDS, and >10% acetonitrile to ensure complete digestion.) Trypsin cleaves the peptide bond between the carboxyl group of arginine or the carboxyl group of lysine and the amino group of the adjacent amino acid. The rate of cleavage occurs more slowly when the lysine and arginine residues are adjacent to acidic amino acids in the sequence or cystine. Cleavage does not occur when lysine or arginine is followed by proline. The sites of trypsin cleavage can be limited to arginine peptide bonds by succinylation. or citraconylation prior to trypsin digestion.
Modification of Lysyl Side Chains Using Citraconic Anhydride: Figure 1.
Authors: Simpson Richard J.
Published: October 2006

INTRODUCTION

Reversible masking of ϵ-amino groups of lysine is a valuable procedure for limiting trypsin hydrolysis of proteins to arginine peptide bonds. Citraconic anhydride is a reversible blocking agent of lysyl side chains. A major advantage of citraconylation is the ease with which the blocking group can be removed under conditions that will not lead to protein denaturation.
Digestion of Insoluble Protein Fractions for Multidimensional Protein Identification Technology (MuDPIT) Analysis
Authors: Schieltz David M. , Washburn Michael P.
Published: October 2006

INTRODUCTION

Proteins associated with insoluble particulate matter from a whole-cell lysate, or carefully prepared membrane samples, can be analyzed using MuDPIT analysis. However, the preliminary steps provided in this protocol are required to prepare a usable complex protein mixture from these types of samples.
Fluorescent Staining of Proteins with SYPRO Ruby
Authors: Simpson Richard J.
Published: October 2006

INTRODUCTION

SYPRO Ruby is a sensitive ruthenium-based fluorescent dye that, similar to silver stains, interacts with basic amino acids, including lysine, arginine, and histidine. Recent studies have shown that this dye is as sensitive as silver staining and completely compatible with matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The staining method has only three steps: fixation, staining, and destaining. Since SYPRO Ruby is very expensive, this dye might be limited to micropreparative gels, as the cost to run the large number of gels done in quantitative studies is prohibitive.
Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy
Published: October 2006

INTRODUCTION

This protocol describes a method for rehydration of IPG gel strips in preparation for their use for isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. Following rehydration, IEF can be performed using either a flatbed unit or a self-contained instrument.
Analysis of Protein Interactions In Vivo with Fluorescence Resonance Energy Transfer (FRET)
Authors: Damelin Marc , Silver Pamela
Published: October 2006

INTRODUCTION

The protocol presented here focuses on a standard microscope-based fluorescence resonance energy transfer (FRET) assay for budding yeast, but many aspects are applicable to other systems and assays. It provides information on experimental design and data analysis procedures that can be extended to FRET experiments in any system, including mammalian cell lines and other model organisms. The steps describing strain construction can be applied to studies in yeast using fluorimetry instead of microscopy. Although the data acquisition and analysis described in this protocol can be a daunting task, careful quantitative analysis is absolutely necessary because of a high degree of cross-talk and many unknown parameters that vary from cell to cell.
Preparation of Vertical SDS Slab Gels: Simultaneous Casting of Multiple-Gradient Gels
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy
Published: October 2006

INTRODUCTION

Gradient SDS-PAGE gels provide the best resolution over a wide range of molecular weights, resulting in sharper protein spots, because diffusion is minimized by the decreasing pore size in the gel. However, gradient gels are more difficult to produce reproducibly; thus, they are commonly cast with multiple-gel casters, which allows for an identical set of gels to be produced for an experiment. Presented here is a method for casting gradient gels using a multiple-gel casting system.
Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy
Published: October 2006

INTRODUCTION

The equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.
EMS Mutagenesis of Arabidopsis Seed
Authors: Weigel Detlef , Glazebrook Jane
Published: October 2006

INTRODUCTION

EMS-mutagenized Arabidopsis seed collections can be purchased from companies or acquired from other researchers. But in some cases (e.g., mutagenesis of a specific genotype), it is necessary for an investigator to generate EMS-mutagenized seed. In this protocol, EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M2 plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype.
Surface Tension of ZrF4‐BaF2‐LaF3 Glass
Authors: BANSAL NAROTTAM P. , DOREMUS ROBERT H.
Published: October 2006

The surface tension of a 62ZrF4.33BaF2.5LaF3 (mol%) glass was measured by a solidified pendant drop technique. The surface tension at 550°C is 0.174 ± 0.005 J/m2.
Fracture of Porous Ceramic Plates under Biaxial Tension‐Compression
Authors: CHEN T.‐J. , KNAPP W. J.
Published: October 2006

The fracture of porous ceramic plates under biaxial tension‐compression was investigated under two different methods of loading. A tensile stress was established in a plate surface by four‐line bending; in addition, a compressive stress was established normal to the tensile stress direction by either of two methods, utilizing (1) a hydraulic jack or (2) filament wrapping. After comparing the experimental results with fracture stresses for uniaxial tension and uniaxial compression, it was concluded that fracture of these porous plates under biaxial tension‐compression was best explained by application of the Griffith theory for cracks of finite sharpness.
The effect of endogenous nitric oxide on mechanical ciliostimulation of human nasal mucosa
Authors: Alberty J. , Stoll W. , Rudack C.
Published: October 2006

Background
Endogenous nitric oxide (NO) production by the inducible NO‐synthase is enhanced in the nasal respiratory epithelium of patients with allergic rhinitis. Recent experimental data suggest endogenous NO to be strongly involved in the regulation of ciliary activity, the driving force of the mucociliary transport system.

Objective
In this study, we investigated the effect of endogenous NO on mechanical stimulation of ciliary activity in a nasal mucosa explant model.

Methods
Cultures of nasal mucosa explants were incubated with TNF‐α and bacterial lipopolysaccharides (LPS) to enhance endogenous NO production. Direct in vitro NO imaging was performed by the fluorescent NO‐indicator DAF‐2 DA and laser scanning confocal microscopy. Ciliary beat frequency (CBF) was determined using a photoelectric technique. Mechanical stimulation was performed by two consecutive flow increments in a closed perfusion chamber. Endogenous NO‐synthesis was blocked by l‐NAME before the second flow stimulation.

Results
Under control conditions the mean rise of CBF relative to baseline was 30.2% during the first flow increment and 30.7% during the second flow increment. Blocking of the endogenous NO synthesis in TNF‐α/LPS‐stimulated cultures reduced baseline CBF by 10.6±2.1% (P<0.05) but the effect of mechanical ciliostimulation on CBF remained unchanged (36.0% vs. 38.2%).

Conclusion
In conclusion, endogenous NO‐ and Ca2+‐dependent mechanical stimulation of ciliary activity probably use independent intracellular signalling pathways. The combination of both effects on ciliary activity is likely to improve the local defence against inhaled allergens in patients with nasal allergies.
Role of intercellular adhesion molecule‐1 in a murine model of toluene diisocyanate‐induced asthma
Authors: Furusho S. , Myou S. , Fujimura M. , Kita T. , Yasui M. , Kasahara K. , Nakao S. , Takehara K. , Sato S.
Published: October 2006

Background
Adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) are thought to contribute to the airway inflammation and airway hyper‐responsiveness (AHR) of allergic asthma. Some differences from allergic asthma have been noted, including airway neutrophilia, and the involvement of ICAM‐1 in toluene diisocyanate (TDI) asthma is currently unclear.

Objective
We utilized mice lacking ICAM‐1 expression (ICAM‐1−/−) to investigate the role of ICAM‐1 in airway inflammation and AHR in TDI‐induced asthma.

Methods
Male C57BL/6J mice (ICAM‐1+/+) and ICAM‐1−/− mice were intranasally sensitized to TDI solution or solvent alone. Airway inflammation, AHR and cytokine secretion were assessed 24 h after challenge by TDI or solvent. The production of antigen‐specific IgG and IgE by TDI sensitized and non‐sensitized mice was determined.

Results
TDI challenge to ICAM‐1+/+ mice induced an increase in airway inflammatory cell numbers, AHR and cytokine secretion of TNF‐α, macrophage inflammatory protein‐2 (MIP‐2), IL‐4, IL‐5 and IFN‐γ into the bronchoalveolar lavage fluid. All these pathophysiological changes were reduced in ICAM‐1−/− mice. Serum levels of TDI‐specific IgG and IgE of ICAM‐1−/− and ICAM‐1+/+ mice were comparable.

Conclusion
These results suggest that ICAM‐1 plays an essential role in airway inflammation and AHR in TDI‐induced asthma.
Changes in Plasma Adrenocorticotropin, Corticosterone, Corticosteroid‐Binding Globulin, and Hippocampal Glucocorticoid Receptor Occupancy/Translocation in Rat Pups in Response to Stress
Authors: Viau Victor , Sharma Shakti , Meaney Michael J.
Published: October 2006

Pituitary‐adrenal responses to stress in the neonatal rat have been reported to be substantially reduced compared to older animals (i.e. a stress hyporesponsive period). This supposed period of endocrine quiescence is characterized by reduced stress‐induced increases in both plasma ACTH and corticosterone. At the same time a number of authors have noted the decreased plasma corticosteroid‐binding globulin (CBG) levels of the neonate, and there is evidence for an increased percentage of free corticosterone as well as age‐related changes in the volume of distribution for corticosterone. These findings suggest that the reduced CBG levels might enhance the biological significance of existing glucocorticoid levels, beyond that assumed on the basis of plasma total corticosterone levels. We examined this question by estimating hippocampal glucocorticoid receptor occupancy and ‘translocation’ in Day 6, Day 15, and adult animals under basal and stressful conditions. The results showed that: 1) plasma ACTH levels were elevated in Day 6 animals in response to acute exposure to ether, maternal separation, and maternal separation + ether, however, ACTH responses were substantially lower than in Day 15 or adult animals; 2) Plasma total corticosterone levels followed a similar pattern; most noteworthy was the potent glucocorticoid response in Day 15 animals to the combination of maternal separation + ether; 3) Plasma CBG levels in Day 6 animals were extremely low (<3% adult values); by Day 15 CBG levels were about 25% of adult levels. Interestingly, maternal separation was associated with a substantial decrease in plasma CBG levels; 4) Hippocampal glucocorticoid receptor occupancy/translocation was similar at all ages under both basal and stress conditions. The only notable exception occurred during maternal separation in Day 15 animals, where the percentage of hippocampal glucocorticoid receptor occupancy/translocation was higher than that observed at any time in either Day 6 or adult animals. This finding is likely related to the decrease in plasma CBG that occurs following separation of Day 15 pups from the dam.

Thus, despite the higher corticosterone level in the adult, the increase in glucocorticoid receptor occupancy/translocation was generally comparable across all ages either under basal conditions, or following stress. These receptor data underscore the importance of developmental changes in plasma CBG levels.
Diuretic and Natriuretic Actions of Melanin Concentrating Hormone in Conscious Sheep
Authors: Parkes David G.
Published: October 2006

Melanin concentrating hormone (MCH) is a 19 amino‐acid peptide expressed in high concentrations within the dorso‐lateral hypothalamus of rats, sheep and man. MCH regulates skin colour and ACTH release in teleost fish, however, its physiological relevance in mammals is unclear. The present study examined the cardiovascular and metabolic actions of intracerebroventricular (i.e.v.) infusion of MCH, and the pro‐MCH derived peptide Neuropeptide‐E‐I (NEI), in conscious, chronically instrumented sheep. Human MCH (1–19) or NEI (1–13) was infused i.e.v. for 24 h into 6 sheep, and measurements were made every 10 min of arterial pressure, heart rate, cardiac output, stroke volume and peripheral blood flow/conductance. Recordings of water intake (H2Oin), urine volume (Uv), urinary Na (UNaV) and K excretion (UKV) were made, as well as hematocrit, plasma Na, K, osmolality, protein, glucose, ACTH, vasopressin, renin, endothelin, ANF, Cortisol and aldosterone concentrations. After 24 h of infusion at 10 μg/h, MCH produced a significant increase in Uv from 0.8 ± 0.2 to 1.4 ± 0.3 l/day, together with an increase in UNaV from 56 ± 8 to 107 ± 14 mmol/day, and in UKV from 202 ± 18 to 369 ± 38 mmol/day. H2Oin was unchanged. Similar renal changes were observed during i.e.v. infusion of NEI. There was no change in any cardiovascular parameter, although hematocrit showed a large decrease with infusion of both peptides after 24 h infusion. Plasma osmolality increased from 291 ±1 to 295 ± 1 mOsm/kg during MCH infusion, whereas total protein and plasma Na and K were unchanged. MCH increased plasma glucose from 3.4 ± 0.2 to 3.8 ± 0.2 mmol/l. Plasma aldosterone exhibited a 30–40% decrease following MCH or NEI infusion, whereas all other plasma concentrations remained unchanged. This study has shown that i.c.v. infusion of MCH or NEI can produce diuretic, natriuretic and kaliuretic changes in conscious sheep, triggered by a possible increase in plasma volume as indicated by the changes in hematocrit. These results, together with anatomical data reporting the presence of MCH/NEI in fluid regulatory areas of the brain, indicate that MCH/NEI may be an important peptide involved in the central control of fluid homeostasis in mammals.
Neurotensin Receptor Down‐Regulation Induced by Dexamethasone and Forskolin in Rat Hypothalamic Cultures is Mediated by Endogenous Neurotensin
Authors: Scarcériaux V. , Souazé F. , Bachelet C. M. , Forgez P. , Bourdel E. , Martinez J. , Rostène W. , Pélaprat D.
Published: October 2006

Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I‐NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high‐affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I‐NT binding sites were present on 3% of the neurons. A 48‐h treatment with forskolin (fsk) decreased 125I‐NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I‐NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I‐NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti‐NT antibody or SR 48692, a non‐peptidic antagonist of NTR, suggesting that the down‐regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist‐induced down‐regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down‐regulate NTR expression via the release of endogenous NT.
11β‐Hydroxysteroid Dehydrogenase Activity in the Hippocampus: Implications for in vivo Corticosterone Receptor Binding and Cell Nuclear Retention
Authors: van Haarst Aernout D. , Welberg Leonie A. M. , Sutanto Win , Oitzl Melly S. , Ron de Kloet E.
Published: October 2006

In this study a possible role of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) in altering the access of corticosteroids to their receptors in the hippocampus is investigated. In vitro, oxidation of corticosterone to 11‐dehydrocorticosterone (11‐DHC) was demonstrated in hippocampal homogenates. Glycyrrhetinic acid (GE) and carbenoxolone (CBX) were potent inhibitors of 11β‐HSD activity and did not display affinity for mineralocorticoid (MRs) nor glucocorticoid receptors (GRs). Intracerebroventricular injection of CBX in vivo (ED50∼30 μg) decreased oxidative activity in hippocampal homogenates, as demonstrated in vitro. In vitro, in hippocampal slices, cell nuclear retention of tritiated corticosterone, but not aldosterone, was markedly enhanced in the presence of GE, which at a concentration of 20 nM was found to inhibit 11β‐HSD activity by about 50% in the intact cell preparation. In contrast to the effect on in vitro cell nuclear uptake, in vivo autoradiography revealed that retention of corticosterone in the hippocampal cell nuclei was not affected after intracerebroventricular treatment with CBX.
Effects of Ovariectomy on GnRH Neuronal Morphology inRhesus Monkey (Macaca mulatta)
Authors: Witkin Joan W.
Published: October 2006

Gonadotropin releasing hormone (GnRH) neurons are typically simple, fusiform cells; however, over the course of prepubertal development increasing numbers take on a ‘spiny’ appearance. Following gonadectomy there is a decrease in the frequency of these spiny GnRH neurons. These observations which were made in the rat suggest that GnRH neurons are directly affected by the gonadal steroid milieu, though they do not themselves contain receptors for these steroidal hormones. In that there are important species differences in the hypothalamic‐pituitary‐gonadal axis between rats and primates, the present study was undertaken to determine whether a reduction in ovarian hormones would produce similar changes in the morphology of GnRH neurons in the monkey. A further aim was to determine whether such changes were localized to a specific brain region.
Prenatal Androgens Defeminize Activation of GnRH Neurons in Response to Estradiol Stimulation
Authors: Wood Ruth I. , Kim Sung‐Ji , Foster Douglas L.
Published: October 2006

To determine if prenatal androgens prevent activation of GnRH neurons in response to estradiol stimulation, Fos colocalization with GnRH was compared in the brains of normal female lambs, normal males, and androgenized females in response to a surge‐inducing dose of estradiol. Blood samples were collected every 1–2 h for 6 h before estradiol treatment up to the time of sacrifice at 17–19 h post‐treatment. Following perfusion, 60 μm coronal brain sections were immunostained for Fos (1:1000, Santa Cruz Biochemicals) and GnRH (1:40,000, LR‐1) using NiCl‐enhanced and unenhanced DAB, respectively. Although LH secretion increased in females before sacrifice, no increase was observed in males or androgenized females. Despite differences in LH secretion, the number and distribution of GnRH neurons was not sexually dimorphic. Moreover, Fos immunostaining was visible throughout steroid‐responsive limbic regions in all three groups of lambs. However, the colocalization of Fos with GnRH was highly sexually dimorphic. In females perfused after the peak of the LH surge, 65.7% of GnRH neurons in the preoptic area, anterior hypothalamus, and mediobasal hypothalamus expressed Fos, whereas only 1.7% of GnRH neurons were Fos‐positive in males and androgenized females. These findings indicate that sex differences in the activation of GnRH neurons in response to estradiol are determined prenatally through the actions of testosterone.
Hydropathy Profiles of Predicted Thyrotropin‐Releasing Hormone Precursors are Highly Conserved Despite Low Similarity of Primary Structures
Authors: Ohide Akira , Ando Hironori , Yanagisawa Tadashi , Urano Akihisa
Published: October 2006

Two types of cDNAs encoding thyrotropin‐releasing hormone (TRH) precursors (TRH‐A andTRH‐B) were amplified from hypothalamic mRNA of sockeye salmon by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The amplification was achieved using two primers which correspond to TRH progenitor sequence (Lys/Arg‐Arg‐Gln‐His‐Pro‐Gly‐Lys/Ag‐Arg). A full length cDNA encoding TRH‐A was obtained by 5′‐ and 3′‐RACE methods. It has a length of 1324 base pairs (bp) that contains sequences of 5′ and 3′ untranslated regions and an open reading frame of 259 codons. The sockeye salmon TRH‐A deduced from the nucleotide sequence tandemly contains 8 copies of TRH progenitor sequences. Another cDNA which encodes a part of TRH‐B consists of 242 bp, and the sequence homology between TRH‐A and ‐B cDNAs is 90%. The result of Southern blot analysis of sockeye and masu salmon genomic DNAs supported the evidence that there are at least two TRH genes in the salmonid. A RT‐PCR analysis of TRH gene expression in various tissues of sockeye salmon showed that strong expression was observed only in the brain. The primary structure of the sockeye salmon TRH‐A shares low similarity to those of human, rat and Xenopus TRH precursors (35, 27 and 44%, respectively). However, their hydropathy profiles were almost the same with each other. The profile of sockeye salmon TRH‐A showed the presence of two discrete hydrophobic regions, one in the N‐terminal region which corresponds to the signal peptide and the other in the C‐terminal region. All of the repetitive TRH progenitor sequences are included in three hydrophilic regions easily recognizable. The present results thus suggest that the three‐dimensional structures of TRH precursors are highly conserved, although the primary structures of TRH precursors have diverged through the evolutionary pathway of vertebrates.
Estradiol, Progesterone, and Prolactin Regulate Maternal Nest‐Building in Rabbits
Authors: González‐Mariscal G. , Melo A. I. , Jiménez P. , Beyer C. , Rosenblatt J. S.
Published: October 2006

Maternal nest‐building in rabbits, expressed across the last third of pregnancy, consists of: digging a burrow, collecting straw and shaping it into a nest inside the burrow, plucking body hair and lining the straw nest with it. The sequential expression of these activities is correlated with specific changes in the plasma concentration of estradiol, progesterone (P), and prolactin (PRL). To further substantiate the participation of these hormones in the control of maternal nest‐building we explored in ovariectomized (ovx) New Zealand white rabbits the capacity of several combinations of such hormones to stimulate digging, straw‐carrying, and hair‐pulling. Does given estradiol benzoate (EB; 5 μg/day from days 3 to 21) plus P (2 or 10 mg/day from days 4 to 16) dug into a substrate from the fourth day of the P treatment until withdrawal of this hormone. The intensity of this effect was greater in the group treated with the high dose of P. Straw‐carrying and hair‐pulling occurred after P withdrawal in a dose‐response way. Food intake, which declines in pregnant females shortly before parturition, decreased to the same extent in both groups of ovx EB‐treated does after P withdrawal. A significant increase in PRL plasma levels was observed on day 9 in does given EB plus 2 mg P/day and at two days following P withdrawal in does given EB plus 10 mg P/day. When such ovx EB/P‐treated does were given bromocriptine to block PRL release (1 or 3 mg/Kg/day, from days 11 to 21) the expression of digging was unmodified. By contrast, bromocriptine abolished the display of straw‐carrying and hair‐pulling, and also prevented the decline in food intake normally following P withdrawal. The addition of ovine PRL to ovx EB/P‐treated does given bromocriptine reduced the expression of digging, did not restore straw‐carrying or hair‐pulling, and provoked a sharp decline in food intake. The possible mechanisms of interaction between PRL and steroid hormones for the regulation of specific aspects of the pregnant doe's physiology and behavior are discussed.
The Relative Roles of the Hypothalamus and Cortisol in the Control of Prolactin Gene Expression in the Anterior Pituitary of the Sheep Fetus
Authors: Phillips I. D. , Fielke S. L. , Young I. R. , McMillen I. C.
Published: October 2006

The neuroendocrine control of prolactin synthesis and secretion before birth is not well understood. We have measured the changes in the level of prolactin mRNA in the anterior pituitary of the fetal sheep throughout the last 15 days of pregnancy (term = 147 ± 3 days gestation). We have also investigated the effects of surgical disconnection of the fetal hypothalamus and pituitary (HPD) with or without long term Cortisol infusion on pituitary prolactin mRNA levels and plasma prolactin concentrations in the late gestation sheep fetus. Prolactin mRNA levels were measured in anterior pituitaries collected from a series of fetal sheep (130–134 days, n = 6; 135–140 days, n = 6; 141–145 days, n = 6) in late gestation. HPD was carried out in ten fetal sheep at 105–115 days gestation and five intact fetal sheep were used as controls. In the HPD group, either saline (HPD + saline group, n = 5) or Cortisol was infused (3.5mg/24 h) for 5 days from 134–136 days gestation (HPD + Cortisol group, n = 5). There was an increase in the ratio of prolactin mRNA:18S rRNA in the fetal pituitary between 130–134 days (0.46 + 0.08, n = 6) and 135–140 days (1.27 ± 0.17 n = 6) which was maintained after 141 days gestation, (1.27 ± 0.11, n = 6). The mean prolactin mRNA: 18 S rRNA ratio was significantly higher (P<0.05) in intact fetal sheep (1.41 ± 0.16, n = 4) than in the HPD fetal sheep after either saline (0.54 ± 0.14, n = 4) or Cortisol (0.74 ± 0.24, n = 5) administration. The mean plasma concentration of prolactin was also higher in the intact group (28.3 ± 3.9 ng/ml) when compared with the HPD + saline group (8.0±3.3 ng/ml) or the HPD+cortisol group (5.6 ± 1.9 ng/ml). We have demonstrated that there is a strong hypothalamic drive to prolactin synthesis and secretion in the fetus and that Cortisol does not act directly at the fetal pituitary to stimulate prolactin synthesis and secretion in late gestation.
Acetylcholine Stimulates Growth Hormone Secretion in the Neonatal Rat Pituitary
Authors: Pintér I. , Makara G. B. , Ács Zs.
Published: October 2006

The direct effect of acetylcholine on pituitary growth hormone secretion during the postnatal period of the rat was studied using a superfusion system. Acetylcholine elicited a dose related stimulatory effect on growth hormone (GH) secretion in the pituitaries from 2‐day old rats. M, muscarinic agonist McN A343 mimicked the GH releasing effect of acetylcholine, nicotine was ineffective. The GH release elicited by acetylcholine diminished with postnatal development, it was small by the end of the third postnatal week and was not demonstrable in the adult pituitaries. The effect of acetylcholine was potently antagonized by pirenzepine (M1 antagonist) and 4‐DAMP (M3 and M1 antagonist) but not by methoctramine (M2 antagonist). It is concluded that unlike in the adult, in the newborn rat the cholinergic regulation of pituitary GH secretion plays a prominent role directly at pituitary level most likely via M1 receptors.
Efficacy of 1.25% and 1% topical cyclosporine in the treatment of severe vernal keratoconjunctivitis in childhood
Authors: Spadavecchia Laura , Fanelli Pietro , Tesse Riccardina , Brunetti Luigia , Cardinale Fabio , Bellizzi Mario , Rizzo Giovanna , Procoli Ugo , Bellizzi Gianfranco , Armenio Lucio
Published: October 2006

Cyclosporine eyedrops 2% have been used for treatment of corticosteroid‐resistant vernal keratoconjunctivitis (VKC) cases. The purpose of our study was to verify the efficacy of 1.25% vs. 1% topical cyclosporine in improving severe form of VKC in childhood. Twenty children with severe VKC, were enrolled in a double‐blind, placebo‐controlled study and received cyclosporine 1.25% in one eye for 2 wk. Then an open trial was conducted during the next 3 months and 2 wk. Thirty‐two more patients were recruited the next year into a new open trial and they received cyclosporine 1% for 4 months. Ocular subjective symptoms and objective signs were scored in all children at entry, 2 wk and 4 months. Skin prick tests and conjunctival scraping tests were also performed; serum immunological and biochemical markers were assessed. The mean score values for severity of subjective symptoms and objective signs were significantly decreased after 2 wk, and 4 months, compared with those at entry (p < 0.001), in both groups of children who received cyclosporine eyedrops 1.25% and 1%, respectively. Serum markers did not differ from the beginning to the end of treatment. Conjunctival eosinophils and cyclosporine serum levels were not detectable at the end of therapy, nor were endothelial corneal cells damaged. Our findings suggest that 1% cyclosporine concentration might be the minimal effective treatment regimen to control symptoms and local inflammation in severe forms of VKC.
Arabidopsis protein repair l‐isoaspartyl methyltransferases: predominant activities at lethal temperatures
Authors: Villa Sarah T. , Xu Qilong , Downie A. Bruce , Clarke Steven G.
Published: October 2006

Protein l‐isoaspartyl (d‐aspartyl) O‐methyltransferases (Enzyme Commission (EC) 2.1.1.77; PIMT or PCMT) are enzymes that initiate the full or partial repair of damaged l‐aspartyl and l‐asparaginyl residues, respectively. These enzymes are found in most organisms and maintain a high degree of sequence conservation. Arabidopsis thaliana (Arabidopsis L. Heynh.) is unique among eukaryotes in that it contains two genes, rather than one, that encode PIMT isozymes. We describe a novel A. thaliana PIMT isozyme, designated AtPIMT2αω, encoded by the PIMT2 gene (At5g50240). We characterized the enzymatic activity of the recombinant AtPIMT2αω in comparison to the other AtPIMT2 isozymes, AtPIMT1, and to the human PCMT1 ortholog, to better understand its role in Arabidopsis. All Arabidopsis PIMT isozymes are active over a relatively wide pH range. For AtPIMT2αω maximal activity is observed at 50°C (a lethal temperature for Arabidopsis); this activity is almost 10 times greater than the activity at the growth temperature of 25°C. Interestingly, enzyme activity decreases after pre‐incubation at temperatures above 30°C. A similar situation is found for the recombinant AtPIMT2ψ and the AtPIMT2ω isozymes, as well as for the AtPIMT1 and human PCMT1 enzymes. These results suggest that the short‐term ability of these methyltransferases to initiate repair under extreme temperature conditions may be a common feature of both the plant and animal species.