bluuebunny/crossref_metadata_2025 huggingface dataset:
An increase in in vivo release of LHRH and precocious puberty by posterior hypothalamic lesions in female rhesus monkeys (Macaca mulatta)
Authors: Windsor-Engnell Bret M. , Kasuya Etsuko , Mizuno Masaharu , Keen Kim L. , Terasawa Ei
Published: December 2006

We have previously shown that a decrease in γ-aminobutyric acid (GABA) tone and a subsequent increase in glutamatergic tone occur in association with the pubertal increase in luteinizing hormone releasing hormone (LHRH) release in primates. To further determine the causal relationship between developmental changes in GABA and glutamate levels and the pubertal increase in LHRH release, we examined monkeys with precocious puberty induced by lesions in the posterior hypothalamus (PH). Six prepubertal female rhesus monkeys (17.4 ± 0.1 mo of age) received lesions in the PH, three prepubertal females (17.5 ± 0.1 mo) received sham lesions, and two females received no treatments. LHRH, GABA, and glutamate levels in the stalk-median eminence before and after lesions were assessed over two 6-h periods (0600–1200 and 1800–2400) using push-pull perfusion. Monkeys with PH lesions exhibited external signs of precocious puberty, including significantly earlier menarche in PH lesion animals (18.8 ± 0.2 mo) than in sham/controls (25.5 ± 0.9 mo, P < 0.001). Moreover, PH lesion animals had elevated LHRH levels and higher evening glutamate levels after lesions, whereas LHRH changes did not occur in sham/controls until later. Changes in GABA release were not discernible, since evening GABA levels already deceased at 18–20 mo of age in both groups and morning levels remained at the prepubertal levels. The age of first ovulation in both groups did not differ. Collectively, PH lesions may not be a good tool to investigate the mechanism of puberty, and, taking into account the recent findings on the role of kisspeptins, the mechanism of the puberty onset in primates is more complex than we initially anticipated.
Endothelin antagonism normalizes VEGF signaling and cardiac function in STZ-induced diabetic rat hearts
Authors: Jesmin Subrina , Zaedi Sohel , Shimojo Nobutake , Iemitsu Motoyuki , Masuzawa Koichi , Yamaguchi Naoto , Mowa Chishimba N. , Maeda Seiji , Hattori Yuichi , Miyauchi Takashi
Published: December 2006

Abnormal alterations in cardiac expression of vascular endothelial growth factor (VEGF) as well as its receptors and impairment in the development of coronary collaterals have recently been reported in diabetic subjects. However, the presence of pharmacological intervention on these defects in diabetes remains unsettled. Here, we studied the effect of endothelin (ET) receptor blockade on cardiac VEGF signaling pathways and cardiac function in Sprague-Dawley rats 5 wk after induction of type I diabetes with streptozotocin (65 mg/kg ip) in comparison with age-matched control rats. After streptozotocin (1 wk), some diabetic rats were treated with the ET receptor antagonist SB-209670 (1 mg/day) for 4 wk. VEGF, its receptors, and its angiogenic signaling molecules [phosphorylated Akt and endothelial nitric-oxide synthase (eNOS)] were analyzed by Western blot, ELISA, real-time PCR, and immunohistochemistry, and cardiac function was evaluated by echocardiography. Coronary capillary morphology was assessed by lectin and enzymatic double staining. We found significant decreases in cardiac expression of VEGF, its receptors, phosphorylation of Akt and eNOS, and coronary capillary density in diabetic rats compared with controls. Treatment of diabetic rats with SB-209670 reversed these alterations to the control levels and ameliorated impairment of cardiac function. From a molecular point of view, the present study is the first to indicate the potential usefulness of an ET receptor antagonist in the treatment of cardiac dysfunction in type I diabetes.
Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function
Authors: Zhong Miao , Parish Bridgette , Murtazina Dilyara A. , Ku Chun-Ying , Sanborn Barbara M.
Published: December 2006

Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V2receptor (V2R) chimera V2ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V2R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V2ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH2-terminal OTR3iC segments in the V2ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19–6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [3H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [3H]OT and the ability to stimulate PI turnover. Affinity for [3H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20–40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.
Muscle growth after postdevelopmental myostatin gene knockout
Authors: Welle Stephen , Bhatt Kirti , Pinkert Carl A. , Tawil Rabi , Thornton Charles A.
Published: December 2006

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50–90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass ∼25% over the following 3 mo in both male and female mice ( P < 0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.
Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line
Authors: Stewart Gavin S. , King Sarah L. , Potter Elizabeth A. , Smith Craig P.
Published: December 2006

Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a ∼40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive 14C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers ( P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea ( P < 0.05, ANOVA) or phloretin ( P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin ( P < 0.05, ANOVA), or intracellular calcium, via ATP ( P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP ( P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.
Direct vasoconstrictor effect of prostaglandin E2on renal interlobular arteries: role of the EP3 receptor
Authors: van Rodijnen William F. , Korstjens Iolente J. , Legerstee Natalee , ter Wee Piet M. , Tangelder Geert-Jan
Published: December 2006

Evidence indicates that prostaglandin E2(PGE2) preferentially affects preglomerular renal vessels. However, whether this is limited to small-caliber arterioles or whether larger vessels farther upstream also respond to PGE2is currently unclear. In the present study, we first investigated the effects of PGE2along the preglomerular vascular tree and subsequently focused on proximal interlobular arteries (ILAs). Proximal ILAs in hydronephrotic rat kidneys as well as isolated vessels from normal kidneys constricted in response to PGE2, both under basal conditions and after the induction of vascular tone. By contrast, smaller vessels, i.e., distal ILAs and afferent arterioles, exhibited PGE2-induced vasodilation. Endothelium removal and pretreatment of single, isolated proximal ILAs with an EP1 receptor blocker (SC51322, 1 μmol/l) or a thromboxane A2receptor blocker (SQ29548, 1 μmol/l) did not prevent vasoconstriction to PGE2. Furthermore, in the presence of SC51322, responses of these vessels to PGE2and the EP1/EP3 agonist sulprostone were superimposable, indicating that PGE2-induced vasoconstriction is mediated by EP3 receptors on smooth muscle cells. Immunohistochemical staining of proximal ILAs confirmed the presence of EP3 receptor protein on these cells and the endothelium. Adding PGE2to normal isolated kidneys induced a biphasic flow response, i.e., an initial flow increase at PGE2concentrations ≤0.1 μmol/l followed by a flow decrease at 1 μmol/l PGE2. Thus our results demonstrate that PGE2affects multiple segments of the preglomerular vascular tree in a different way. At the level of the proximal ILAs, PGE2had a direct vasoconstrictor action mediated by EP3 receptors.
Intercellular calcium signaling and nitric oxide feedback during constriction of rabbit renal afferent arterioles
Authors: Uhrenholt T. R. , Schjerning J. , Vanhoutte P. M. , Jensen B. L. , Skøtt O.
Published: December 2006

Vasoconstriction and increase in the intracellular calcium concentration ([Ca2+]i) of vascular smooth muscle cells may cause an increase of endothelial cell [Ca2+]i, which, in turn, augments nitric oxide (NO) production and inhibits smooth muscle cell contraction. This hypothesis was tested in microperfused rabbit renal afferent arterioles, using fluorescence imaging microscopy with the calcium-sensitive dye fura-2 and the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorescein. Both dyes were loaded into smooth muscle and endothelium. Depolarization with 100 mmol/l KCl led to a transient vasoconstriction which was converted into a sustained response by N-nitro-l-arginine methyl ester (l-NAME). Depolarization increased smooth muscle cell [Ca2+]ifrom 162 ± 15 nmol/l to a peak of 555 ± 70 nmol/l ( n = 7), and this response was inhibited by 80% by the l-type calcium channel blocker calciseptine. After a delay of 10 s, [Ca2+]iincreased in endothelial cells immediately adjacent to reactive smooth muscle cells, and this calcium wave spread in a nonregenerative fashion laterally into the endothelial cell layer with a velocity of 1.2 μm/s. Depolarization with 100 mmol/l KCl led to a significant increase in NO production ([NO]i) which was inhibited by l-NAME ( n = 5). Acetylcholine caused a rapid increase in endothelial [Ca2+]i, which did not transfer to the smooth muscle cells. l-NAME treatment did not affect changes in smooth muscle [Ca2+]iafter depolarization, but it did increase the calcium sensitivity of the contractile apparatus. We conclude that depolarization increases smooth muscle [Ca2+]iwhich is transferred to the endothelial cells and stimulates NO production which curtails vasoconstriction by reducing the calcium sensitivity of the contractile apparatus.
Proteomic identification of proteins associated with the osmoregulatory transcription factor TonEBP/OREBP: functional effects of Hsp90 and PARP-1
Authors: Chen Ye , Schnetz Michael P. , Irarrazabal Carlos E. , Shen Rong-Fong , Williams Chester K. , Burg Maurice B. , Ferraris Joan D.
Published: December 2006

Hypertonicity (e.g., high NaCl) activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1-547 of TonEBP/OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/OREBP. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90β and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we find that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl.
Modulation of Ca2+ Channels by Heterologously Expressed Wild-Type and Mutant Human μ-Opioid Receptors (hMORs) Containing the A118G Single-Nucleotide Polymorphism
Authors: Margas Wojciech , Zubkoff Ira , Schuler H. Gregg , Janicki Piotr K. , Ruiz-Velasco Victor
Published: December 2006

The most common single-nucleotide polymorphism (SNP) of the human μ-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca2+ channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca2+ channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration–response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC50 values (nM) in cells expressing wild-type hMOR was: DAMGO (31) ≫ morphine (76) ≅ M-6-G (77) ≅ endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) ≫ morphine (39) ≫ endomorphin I (74) ≅ M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca2+ current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.
Insights into the immune transcriptome of the shrimpLitopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge
Authors: Robalino Javier , Almeida Jonas S. , McKillen David , Colglazier Joan , Trent Harold F. , Chen Yian Ann , Peck Megan E. T. , Browdy Craig L. , Chapman Robert W. , Warr Gregory W. , Gross Paul S.
Published: December 2006

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described. The unigenes printed on the microarray were derived from the analyses of 7,021 expressed sequence tags obtained from standard cDNA libraries as well as from libraries generated by suppression subtractive hybridization, after challenging shrimp with a variety of immune stimuli. The general utility of the cDNA microarray was demonstrated by interrogating the array with labeled RNA from four different shrimp tissues (gills, hemocytes, hepatopancreas, and muscle) and by analyzing the transcriptomic response of shrimp to a lethal challenge with white spot syndrome virus. Our results indicate that white spot syndrome virus infection upregulates (in the hepatopancreas) genes encoding known and potential antimicrobial effectors, while some genes involved in protection from oxidative stress were found to be downregulated by the virus.
Serine threonine protein kinases of mycobacterial genus: phylogeny to function
Authors: Narayan Azeet , Sachdeva Preeti , Sharma Kirti , Saini Adesh K. , Tyagi Anil K. , Singh Yogendra
Published: December 2006

Serine/threonine protein kinases (STPKs) are known to act as sensors of environmental signals that thereby regulate developmental changes and host pathogen interactions. In this study, we carried out comparative genome analysis of six completely sequenced pathogenic and nonpathogenic mycobacterial species to systematically characterize the STPK complement of mycobacterium. Our analysis revealed that while Mycobacterium tuberculosis strains have 11 conserved kinases, this number varies from 4 to 24 in other mycobacterial species. pknA, an essential STPK encoding gene, was found to be truncated in the initial analysis of M. avium subsp. paratuberculosis (Map) and M. tuberculosis C genomes. However, resequencing of pknA gene in Map confirmed that the truncation was due to a sequencing error. The conservation of division and cell wall gene cluster involved in cell envelope biosynthesis and cell division, in the vicinity of pknL locus, implicates a possible role of PknL in cell division and envelop biosynthesis. We identified a cyclophilin domain as part of a mycobacterial kinase in Map that suggests a plausible regulation of cyclophilins by phosphorylation. The co-inheritance of pknA, pknB, pknG, and pknL loci across genomes and some unique repertoire of pathogen-specific kinases such as pknI and pknJ of Mtb complex suggest similitude and divergence between pathogenic and nonpathogenic signaling. This study would add another dimension toward identification of physiological substrates and thereby function, while resolving the existing complexities in signaling network between the two domains of life, pathogen and nonpathogen.
Novel in vivo imaging shows up-regulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies
Authors: Gong Jing , Yang David , Kohanim Saady , Humphreys Robin , Broemeling Lyle , Kurzrock Razelle
Published: December 2006

Susceptibility to apoptosis by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is mediated through cognate death receptor signaling. We hypothesized that auto-amplification of this apparatus would enhance antitumor effects in vivo and could be optimized using the results obtained from novel imaging techniques. We therefore imaged mice bearing human colorectal cancer (Colo205) tumor xenografts with HGS-ETR1 and HGS-ETR2 agonist antibodies to TRAIL receptor-1 (TRAIL-R1) and TRAIL-R2, respectively, after radiolabeling the antibodies. Paclitaxel significantly increased in vivo expression of TRAIL-R1 and TRAIL-R2 in a time-dependent manner. The imaging results were confirmed by immunoblots for steady-state protein levels (>20-fold increase in TRAIL-R1 and TRAIL-R2 levels in tumor xenografts by 48 h after paclitaxel administration). TRAIL-R1 and TRAIL-R2 mRNA expression did not change, suggesting that these effects were posttranscriptional. Sequential treatment with paclitaxel followed by HGS-ETR1 or HGS-ETR2 after 48 h resulted in markedly enhanced antitumor activity against Colo205 mouse xenografts. Our experiments suggest that sequential taxane treatment followed by TRAIL-R agonist antibodies could be applied in the clinic, and that novel imaging techniques using radiolabeled receptor antibodies may be exploitable to optimize sequence timing and patient selection. [Mol Cancer Ther 2006;5(12):2991–3000]
Identification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib
Authors: Serrels Alan , Macpherson Iain R.J. , Evans T.R. Jeffry , Lee Francis Y. , Clark Edwin A. , Sansom Owen J. , Ashton Gabrielle H. , Frame Margaret C. , Brunton Valerie G.
Published: December 2006

Elevated levels of Src kinase expression have been found in a variety of human epithelial cancers. Most notably in colon cancer, elevated Src expression correlates with malignant potential and is also associated with metastatic disease. Dasatinib (BMS-354825) is a novel, orally active, multi-targeted kinase inhibitor that targets Src family kinases and is currently under clinical evaluation for the treatment of solid tumors. However, the effects of dasatinib on epithelial tumors are not fully understood. We show that concentrations of dasatinib that inhibit Src activity do not inhibit proliferation in 10 of 12 colon cancer cells lines. However, inhibition of integrin-dependent adhesion and migration by dasatinib correlated with inhibition of Src activity, suggesting that dasatinib may have anti-invasive or anti-metastatic activity and antiproliferative activity in epithelial tumors. Using phospho-specific antibodies, we show that inhibition of Src activity in colon cancer cell lines correlates with reduced phosphorylation of focal adhesion kinase and paxillin on specific Src-dependent phosphorylation sites. We have validated the use of phospho-specific antibodies against Src Tyr419 and paxillin Tyr118 as biomarkers of dasatinib activity in vivo. Colon carcinoma–bearing mice treated with dasatinib showed a decrease in both phospho-Src Tyr419 and phospho-paxillin Tyr118 in peripheral blood mononuclear cells, which correlated with inhibition of Src activity in the colon tumors. Thus, peripheral blood mononuclear cells may provide a useful surrogate tissue for biomarker studies with dasatinib using inhibition of Src Tyr419 and paxillin Tyr118 phosphorylation as read-outs of Src activity. [Mol Cancer Ther 2006;5(12):3014–22]
The synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole blocks nuclear factor-κB activation through direct inhibition of IκB kinase β
Authors: Yore Mark M. , Liby Karen T. , Honda Tadashi , Gribble Gordon W. , Sporn Michael B.
Published: December 2006

The synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) is a multifunctional agent with potent anti-inflammatory, antiproliferative, cytoprotective, and apoptotic activities, whose molecular targets are unknown. Using both cell-free and cellular assays, we show that CDDO-Im is a direct inhibitor of IκB kinase (IKK) β and that it thereby inhibits binding of nuclear factor-κB to DNA and subsequent transcriptional activation. Pretreatment of cells with CDDO-Im prevents IκBα phosphorylation and degradation in response to tumor necrosis factor α. The kinetics of this inhibition by CDDO-Im are rapid and occur within 15 min. A biotinylated analogue of CDDO-Im showed that CDDO-Im binds to the IKK signalsome. Furthermore, we show that Cys179 on IKK is a target for CDDO-Im. This is the first report to show that this novel synthetic triterpenoid binds to and inhibits IKKβ directly. [Mol Cancer Ther 2006;5(12):3232–9]
Expression of SDF-1 and CXCR4 during Reorganization of the Postnatal Dentate Gyrus
Authors: Berger Omri , Li Guangnan , Han Szu-Min , Paredes Mercedes , Pleasure Samuel J.
Published: December 2006

Previous studies have demonstrated that stromal cell-derived factor 1 (SDF-1) is crucial for early dentate development; however, the mouse mutants for this chemokine and its only receptor, CXCR4, are neonatally lethal, making conclusions about the role of these molecules in postnatal development difficult to sustain. Previous expression analyses have used single labeling, but the distribution of CXCR4 is complex and to determine the cell types expressing CXCR4 requires multiple marker labeling. In this study, we examined the distribution of SDF-1 and CXCR4 mRNAs during the first postnatal weeks, combining these markers with several other cell-type-specific markers. We found that SDF-1 has three sites of expression: (1) continuation of prenatal expression in the meninges; (2) expression in Cajal-Retzius cells occupying the molecular layer of the upper and lower blades of the dentate, and (3) the maturing dentate granule neurons themselves. The timing of expression in these three sites corresponds to alterations in the distribution of the primary cell types expressing CXCR4 during the same periods, notably the expression of CXCR4 in radial-glial-like GFAP-expressing dentate precursors and immature dentate granule neurons. Taken together, our data suggest potential ongoing roles for SDF-1/CXCR4 signaling in the dentate gyrus during the early postnatal period that will be tested in the future with more precise genetic approaches.
Anatomic and Electrophysiologic Evidence for a Proconvulsive Circuit in the Dentate Gyrus of Reeler Mutant Mice, an Animal Model of Diffuse Cortical Malformation
Authors: Patrylo Peter R. , Willingham Amy
Published: December 2006

Although cortical malformations (CMs) are often associated with epilepsy, the underlying mechanisms are unknown. The reeler mouse is a model of CM with enhanced susceptibility to epileptiform activity, including the in vitro dentate gyrus, a region normally resistant to seizures. In this study, field potential recordings in hippocampal slices and the Timm stain were used to examine mossy fiber distribution in the dentate gyrus. In artificial cerebrospinal fluid containing bicuculline, 100% of reeler slices and 0% of control slices had spontaneous and antidromic evoked prolonged negative field potential shifts that were blocked by glutamate receptor antagonists. Sections from reeler mice, but not controls, exhibited a dark band of Timm’s stain at the molecular layer/granule cell layer border. These data reveal that mossy fiber distribution is altered in reeler mice and coincides with the presence of an abnormal proconvulsive glutamatergic circuit.
Balance between Neurogenesis and Gliogenesis in the Adult Hippocampus: Role for Reelin
Authors: Zhao Shanting , Chai Xuejun , Frotscher Michael
Published: December 2006

The extracellular matrix protein reelin is essential for the proper radial migration of cortical neurons. In reeler mice lacking reelin, there is a malformation of the radial glial scaffold required for granule cell migration. Immunostaining for glial fibrillary acidic protein (GFAP) reveals abundant radial glial cells with long fibers traversing the granular layer in the wild type, but almost exclusively astrocytes in the reeler mutant. With the concept that radial glial cells are precursors of neurons, we hypothesized that the balance between neurogenesis and gliogenesis is altered in the reeler mutant. To this end, adult reeler mutants and their wild-type littermates were injected with bromodeoxyuridine (BrdU), a marker of newly generated cells. When compared to wild-type animals, we found a reduction in the number of BrdU-labeled cells in the adult reeler dentate gyrus. Moreover, whereas there was a dramatic decrease in the number of newly generated granule cells identified by double labeling for BrdU and NeuN, the number of BrdU-labeled, GFAP-positive astrocytes had increased. Decreased neurogenesis in the adult reeler dentate gyrus was confirmed by immunostaining for doublecortin, a marker of newly generated neurons. These results indicate that adult neurogenesis is altered in the reeler dentate gyrus and that newly generated cells preferentially differentiate into astrocytes.
Dentate Development in Organotypic Hippocampal Slice Cultures from p35 Knockout Mice
Authors: Wenzel H. Jürgen , Tamse Catherine T. , Schwartzkroin Philip A.
Published: December 2006

Abnormal brain development, induced by genetic influences or resulting from a perinatal trauma, has been recognized as a cause of seizure disorders. To understand how and when these structural abnormalities form, and how they are involved in epileptogenesis, it is important to generate and investigate animal models. We have studied one such model, a mouse in which deletion of the p35 gene (p35–/–) gives rise to both structural disorganization and seizure-like function. We now report that aberrant dentate development can be recognized in the organotypic hippocampal slice culture preparation generated from p35–/– mouse pups. In these p35–/– cultures, an abnormally high proportion of dentate granule cells migrates into the hilus and molecular layer, and develops aberrant dendritic and axonal morphology. In addition, astrocyte formation in the dentate gyrus is disturbed, as is the distribution of GABAergic interneurons. Although the p35–/– brain shows widespread abnormalities, the disorganization of the hippocampal dentate region is particularly intriguing since a similar pathology is often found in hippocampi of temporal lobe epilepsy patients. The abnormal granule cell features occur early in development, and are independent of seizure activity. Further, these aberrant patterns and histopathological features of p35–/– culture preparations closely resemble those observed in p35 knockout mice in vivo. This culture preparation thus provides an experimentally accessible window for studying abnormal developmental factors that can result in seizure propensity.
Pneumococcal Meningitis Induces Apoptosis in Recently Postmitotic Immature Neurons in the Dentate Gyrus of Neonatal Rats
Authors: Grandgirard D. , Bifrare Y.-D. , Pleasure S.J. , Kummer J. , Leib S.L. , Täuber M.G.
Published: December 2006

Bacterial meningitis is associated with high rates of morbidity and mortality, despite advances in antibiotic therapy. Meningitis caused by Streptococcus pneumoniae is associated with a particularly high incidence of neurological sequelae including deficits resulting from damage to the hippocampus. Previous studies have documented that in neonatal rats with experimental pneumococcal meningitis, cells in the subgranular layer of the dentate gyrus undergo apoptosis. The aim of the present study was to define in more detail the nature of the dying cells in the dentate gyrus. Using bromodeoxyuridine labeling at different times before infection combined with immunocytochemistry, we identified the vulnerable cells as those which underwent mitosis 6–10 days before infection. A majority of these cells are of neuronal lineage. Thus, immature neuronal cells several days after the last cell division are preferentially triggered into apoptosis during pneumococcal meningitis. The loss of these cells may contribute to the long-lasting impairment of hippocampal function identified in animal models and in humans after bacterial meningitis.
Loss of Embryonic MET Signaling Alters Profiles of Hippocampal Interneurons
Authors: Martins Gabriela J. , Plachez Céline , Powell Elizabeth M.
Published: December 2006

Hippocampal interneurons arise in the ventral forebrain and migrate dorsally in response to cues, including hepatocyte growth factor/scatter factor which signals via its receptor MET. Examination of the hippocampus in adult mice in which MET had been inactivated in the embryonic proliferative zones showed an increase in parvalbumin-expressing cells in the dentate gyrus, but a loss of these cells in the CA3 region. An overall loss of calretinin-expressing cells was seen throughout the hippocampus. A similar CA3 deficit of parvalbumin and calretinin cells was observed when MET was eliminated only in postmitotic cells. These data suggest that MET is required for the proper hippocampal development, and embryonic perturbations lead to long-term anatomical defects with possible learning and memory dysfunction.
Persistently Decreased Basal Synaptic Inhibition of Hippocampal CA1 Pyramidal Neurons after Neonatal Hypoxia-Induced Seizures
Authors: Sanchez Russell M. , Justice Jason A. , Zhang Kun
Published: December 2006

Hypoxia is the most common cause of neonatal seizures and can lead to epilepsy, but the epileptogenic mechanisms are not yet understood. We have previously shown that hypoxia-induced seizures in the neonatal rat result in acutely decreased amplitudes and frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons. In the current study, we asked whether such changes persist for several days following hypoxia-induced seizures. Similar to the acute findings, we observed decreased frequency and amplitudes of sIPSCs and decreased mIPSC amplitudes in CA1 pyramidal neurons at 3–5 days after hypoxia. However, in contrast to the acute findings, we observed no differences between hypoxia-treated and control groups in mIPSC frequency. Additionally, by 7 days after hypoxia, sIPSC amplitudes in the hypoxia group had recovered to control levels, but sIPSC frequency remained decreased. These data indicate that the persistently decreased sIPSC frequency result from decreased firing of presynaptic inhibitory interneurons, with only transient possible changes in postsynaptic responses to GABA release.
Recurrent Seizures and the Molecular Maturation of Hippocampal and Neocortical Glutamatergic Synapses
Authors: Swann John W. , Le John T. , Lee Chong L.
Published: December 2006

Recurrent seizures in animal models of early-onset epilepsy have been shown to produce deficits in spatial learning and memory. While neuronal loss does not appear to underlie these effects, dendritic spine loss has been shown to occur. In experiments reported here, seizures induced either by tetanus toxin or flurothyl during the second postnatal week were found to reduce the expression of NMDA receptor subunits in both the hippocampus and neocortex. Most experiments focused on alterations in the expression of the NR2A subunit and its associated scaffolding protein, PSD95, since their expression is developmentally regulated. Results suggest that the depression in expression can be delayed by at least 5 days but persists for at least 3–4 weeks. These effects were dependent on the number of seizures experienced, and were not observed when seizures were induced in adult mice. Taken together, the results suggest that recurrent seizures in infancy may interrupt synapse maturation and produce persistent decreases in molecular markers for glutamatergic synapses – particularly components of the NMDA receptor complex implicated in learning and memory.
Postnatal Development of the Primate Hippocampal Formation
Authors: Lavenex Pierre , Banta Lavenex Pamela , Amaral David G.
Published: December 2006

The hippocampal formation is a multicomponent region of the medial temporal lobe preferentially involved in declarative and relational memory processing. Behavioral studies have suggested a protracted functional maturation of these structures in primates, and postnatal developmental abnormalities in the hippocampal formation are thought to contribute to neurodevelopmental disorders, such as autism, schizophrenia, epilepsy and Down syndrome. Despite all that we know about the functional organization of the adult hippocampal formation, notably absent is a systematic study of its postnatal maturation in primates. In this article, we review current knowledge of the structural development of the primate hippocampal formation and present new data on its postnatal neuroanatomical development. We summarize what is known about the neurobiological processes, such as the addition of new neurons, the establishment and elaboration of connectivity, and the neurochemical changes, that underlie the structural development and functional maturation of the primate hippocampal formation. We conclude that there is yet insufficient information to identify distinct developmental windows during which different hippocampal regions undergo specific maturational processes. For this reason, it is currently impossible to determine the ages at which specific hippocampal circuits become structurally mature and potentially capable of supporting defined, age-specific functional processes. Together with work in rodents, systematic studies of the structural development and functional maturation of the monkey hippocampal formation will be necessary to gain insight not only into the types of information processing that it subserves, but also into the specific maturational processes that might be affected in human neurodevelopmental disorders.
Meiosis Onset Is Postponed to Postnatal Stages during Ovotestis Development in Female Moles
Authors: Zurita F. , Carmona F.D. , Lupiáñez D.G. , Barrionuevo F.J. , Guioli S. , Burgos M. , Jiménez R.
Published: December 2006

In mammals, germ cells are important both during development and for the function of female gonads, whereas male gonads may develop in the absence of germ cells. The gonads of female moles (genus Talpa) develop according to a testis-like pattern which results in the formation of ovotestes. In this paper, we studied the expression pattern of several pre-meiotic and meiotic germ cell markers, in order to establish the precise time of meiosis onset in the mole species T. occidentalis, and to investigate the location and possible role of germ cells in ovotestis organogenesis. Our results evidenced that: (1) the asymmetrical distribution of primordial germ cells, which concentrate in the cortex of the XX gonad, is brought about by germ cell depletion from the medulla between the s5a and s5b stages, (2) XX germ cells enter meiosis postnatally, which is quite exceptional among eutherian mammals, and (3) XX but not XY germ cells of moles express DMRT1 during premeiotic stages of development, an expression pattern not described previously in vertebrates.
Recombinant Human Erythropoietin Is Associated with Increased Overall Survival in Patients with Multiple Myeloma
Authors: Baz R. , Walker E. , Choueiri T.K. , Abou Jawde R. , Brand C. , McGowan B. , Yiannaki E. , Andresen S. , Hussein M.A.
Published: December 2006

Background: Recombinant human erythropoietin (rhEPO) is effective for the treatment of anemia associated with multiple myeloma. Data from animal studies and case reports suggest that rhEPO has antineoplastic properties. Methods: Two hundred and ninety-two patients enrolled on different chemotherapy clinical trials at the Cleveland Clinic Myeloma Program between 1997 and 2003 were the subjects of this study. Information on erythropoietin use as well as baseline prognostic variables were collected retrospectively. Results: The population consisted of 257 patients with multiple myeloma treated at the Cleveland Clinic Foundation from 1997 to 2003 and followed for at least 1 month. Thirty-five patients were excluded from this analysis because information on erythropoietin use was not available. One hundred and twenty-seven patients received rhEPO for at least 1 month and the rest did not received rhEPO. On average, patients who received rhEPO were older, had a higher Southwest Oncology Group (SWOG) stage, higher serum creatinine, lower serum hemoglobin, higher β2-microglobulin, lower platelet counts, and a longer time from diagnosis to enrollment at the myeloma program (p < 0.001 for all). After adjusting for age, months from diagnosis to enrollment, serum creatinine, hemoglobin, platelet count, and β2-microglobulin, the use of rhEPO was associated with improved overall survival (hazard ratio = 0.6; 95% CI = 0.38–0.94) in patients with SWOG stages II, III and IV but not in patients with SWOG stage I. Conclusion: rhEPO was associated with improved overall survival in this population of anemic multiple myeloma patients with SWOG stages of II, III and IV. A prospective randomized trial is warranted to corroborate this finding.
Everolimus Is a Potent Inducer of Erythroid Differentiation and γ-Globin Gene Expression in Human Erythroid Cells
Authors: Zuccato Cristina , Bianchi Nicoletta , Borgatti Monica , Lampronti Ilaria , Massei Francesco , Favre Claudio , Gambari Roberto
Published: December 2006

We studied the effects of everolimus on the erythroid differentiation of human leukaemic K562 cells and on the cultures of erythroid progenitors derived from the peripheral blood of β-thalassaemia patients. A quantitative real-time reverse-transcription polymerase chain reaction assay was employed for the quantification of the accumulation of globin mRNAs. The results obtained demonstrate that everolimus is a potent inducer of the erythroid differentiation of K562 cells. Erythroid induction is associated with an increase in α- and γ-globin mRNAs. In erythroid precursor cells from 4 β-thalassaemia patients, everolimus stimulated a preferential increase (ranging from 1.8- to 7.2-fold) in γ-globin mRNA. Only minor effects were observed on the expression of α-globin genes. These results, in our opinion, are of interest as this compound is already employed in clinical trials as an anti-rejection agent following kidney transplantation. These data suggest that everolimus warrants further evaluation as a potential therapeutic drug in the treatment of β-thalassaemia.
Insufficient Arteriovenous Fistulae in Hemodialysis Patients
Authors: Janzen J. , Mickley V.
Published: December 2006

Background: For an accurate assessment of structural changes in arteriovenous fistulae (AVF) a microscopic analysis is mandatory. Methods: 25 insufficient AVF were analyzed with the light microscope using standard histological and immunohistochemical techniques. Results: In 7 patients (28%) atherosclerotic lesions as causes of AVF insufficiency were found. The other 18 patients (72%) had an inhomogeneous spectrum of nonatherosclerotic lesions, for instance intimal hyperplasia. Conclusions: Histopathologic analysis of insufficient AVF helps clarify the underlying changes in the structure of the vessel wall.
Genetic Analysis of Gitelman Syndrome Patients from the Czech Republic and Slovakia – Three Novel Mutations Found
Authors: Urbanová Markéta , Reiterová Jana , Ryšavá Romana , Štekrová Jitka , Merta Miroslav
Published: December 2006

Background: To investigate the genetic cause of inherited hypokalemic metabolic alkalosis associated with Gitelman’s syndrome, we searched for mutations in the SLC12A3 gene (thiazide-sensitive NaCl cotransporter) among a set of patients from the Czech Republic and Slovakia. Methods: We collected blood samples of patients from 16 families with characteristic clinical features. DNA was analyzed for mutation detection with SSCP and subsequent sequencing. Several mutations might be missed when only the SSCP method is applied, therefore direct sequencing of all the 26 exons became an essential tool. Results: Genetic analysis revealed mainly missense mutations. Two novel mutations, c.480dupC (p.Pro160fsX97) and c.238dupCC (p. Pro79fsX35), caused a frameshift and preliminary stop codon appearance. Missense mutation c.790 G → C (p.Gly264Arg) has never been reported before. Mutation c.1315G → A (p.Gly439Ser) was frequent among our collection of unrelated patients (5 out of 16). Homozygous Gly439Ser was observed in a patient with chondrocalcinosis. Conclusion: We identified 13 different causative mutations in a cohort of Gitelman syndrome patients. Three of those mutations are novel. The occurrence of two mutation detections per individual corresponding to a recessive trait of inheritance was 62.5%. Gly439Ser is the most frequent type of mutation among our patients. Statistic evaluation of genotype/phenotype correlation could not be carried out.
Neuropsychological Comparison of Alzheimer’s Disease and Dementia with Lewy Bodies
Authors: Crowell Timothy A. , Luis Cheryl A. , Cox David E. , Mullan Michael
Published: December 2006

Background/Aims: The present study examined the patterns of memory and cognitive performance associated with Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). Methods: A battery of standardized neuropsychological tests was administered to individuals with these disorders as well as to a group of cognitively intact controls. The battery included measures of memory (learning, recall and recognition), language, visuospatial ability, psychomotor speed, executive functioning and mood. All subjects (n = 115) were evaluated at a memory disorder clinic and were diagnosed based on published criteria. Results: The controls outperformed both dementia groups on all cognitive measures. With respect to memory, the DLB group scored significantly higher than the AD group on measures of word list free recall and recognition (p ≤ 0.001). In other cognitive domains, the AD group performed significantly better than the DLB group on constructional praxis, sustained attention, phonemic fluency, spatial judgment, psychomotor speed and working memory (all p ≤ 0.01). Conclusion: These findings support the usefulness of memory and other cognitive test score patterns as in distinguishing AD from DLB, particularly in mild to moderately demented populations that may not present with hallmark symptomology.