Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.     

Day-night variation in the in vitro contractility of aorta and mesenteric and renal arteries in transgenic hypertensive rats

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Synthesis,characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.     

Genetic susceptibility to keloid disease and hypertrophic scarring: transforming growth factor beta1 common polymorphisms and plasma levels

Keloid disease and hypertrophic scars are dermal tumors that are often familial and typically occur in certain races. Their exact etiology is still unknown. Transforming growth factor beta1 (TGF-beta1) plays a central role in wound healing and fibrosis and has been implicated in the pathogenesis of keloid disease and hypertrophic scar. The aims of this study were to measure the plasma level of TGF-beta1 in patients compared with controls, and to investigate the association of five common single nucleotide polymorphisms in TGF-beta1 with the risk of keloid disease and hypertrophic scar formation. Platelet-poor plasma levels of TGF-beta1 in 60 patients (15 with hypertrophic scar and 45 with keloid disease) and 18 controls were measured using an enzyme-linked immunoabsorbent assay technique. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-beta1 polymorphisms. DNA samples from 133 patients (101 with keloid disease and 32 with hypertrophic scar) and 200 controls were examined. All patients and controls were Caucasians of Northern European extraction. There was no statistically significant difference in TGF-beta1 plasma levels between patients with keloid disease and hypertrophic scar and controls. There was also no statistically significant difference in genotype or allele frequency distributions between patients and controls for codons 10, 25, and 263 and for -509 and -800 single nucleotide polymorphisms of the TGF-beta1 gene. These results suggest that TGF-beta1 plasma levels and common polymorphisms are not associated with a risk of keloid disease and hypertrophic scar formation. This lack of association may be significant in view of the importance attached to the role of TGF-beta1 in dermal scarring. To the authors' knowledge, this is the first report of a case-control association study in keloid disease and hypertrophic scars using any single nucleotide polymorphisms.     

Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP

The cellular prion protein (PrP^C) consists of a flexible N-terminal tail (FT, aa 23–128) hinged to a membrane-anchored globular domain (GD, aa 129–231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrP_Δ141–225, or “FTgpi”). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.     

Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts

Cadherins are the most crucial membrane proteins for the formation of tight and compact cell-cell contacts. Cadherin-based cell-cell adhesions are dynamically established and/or disrupted during various physiological and pathological processes. However, the molecular mechanisms that regulate cell-cell contacts are not fully understood. In this paper, we report a novel functional role of casein kinase 1 (CK1) in the regulation of cell-cell contacts. Firstly, we observed that IC261, a specific inhibitor of CK1, stabilizes cadherin-based cell-cell contacts, whereas the overexpression of CK1 disrupts them. CK1 colocalizes with E-cadherin and phosphorylates the cytoplasmic domain of E-cadherin in vitro and in a cell culture system. We show that the major CK1 phosphorylation site of E-cadherin is serine 846, a highly conserved residue between classical cadherins. Constitutively phosphorylated E-cadherin (S846D) is unable to localize at cell-cell contacts and has decreased adhesive activity. Furthermore, phosphorylated E-cadherin (S846D) has weaker interactions with beta-catenin and is internalized more efficiently than wild-type E-cadherin. These data indicate that CK1 is a novel negative regulator of cadherin-based cell-cell contacts.     

Age and sex differentially affect regional changes in one repetition maximum strength

To assess the influences of age and sex on regional changes in 1 repetition maximum (1RM) strength, 10 young men (20-30 years), 8 young women (20-30 years), 11 older men (65- 75 years), and 10 older women (65-75 years) were studied before and after a 24-week whole-body strength training program. Changes in 1RM strength were analyzed for each individual exercise, as well as by calculating a total body score (TBS), an upper body score (UBS), and a lower body score (LBS). The effect of age and sex on changes in 1RM strength was analyzed using a repeated measures analysis of variance. When changes in strength for individual exercises were analyzed, the chest press, lat pulldown, shoulder press, and triceps pushdown were affected by both age (p < 0.05) and sex (p < 0.05), while the biceps curls were only influenced by age (p < 0.05). For the lower body, the leg press changes in 1RM strength were influenced by age (p < 0.0001), while leg extension was influenced by sex (p < 0.05). Total body score, UBS, and LBS showed significant increases with 24 weeks of ST (p < 0.001, all). Changes in TBS and UBS were affected by age (p < 0.001, both) and sex (p < 0.05 and p < 0.001, respectively). Younger subjects showed a greater increase in strength than older subjects, and men showed a greater increase in strength compared with women. Changes in LBS were affected by age (p < 0.001), with younger subjects showing a greater increase in strength compared with the older subjects, but not by sex (p = 0.464). These data indicate that regional increases in strength are differentially affected by age and sex.     

Circadian periodicity of cerebral blood flow revealed by laser-Doppler flowmetry in awake rats: relation to blood pressure and activity

Cardiovascular parameters such as arterial blood pressure (ABP) and heart rate display pronounced circadian variation. The present study was performed to detect whether there is a circadian periodicity in the regulation of cerebral perfusion. Normotensive Sprague-Dawley rats (SDR, approximately 15 wk old) and hypertensive (mREN2)27 transgenic rats (TGR, approximately 12 wk old) were instrumented in the abdominal aorta with a blood pressure sensor coupled to a telemetry system for continuous recording of ABP, heart rate, and locomotor activity. After 5-12 days, a laser-Doppler flow (LDF) probe was attached to the skull by means of a guiding device to measure changes in brain cortical blood flow (CBF). After the animals recovered from anesthesia, measurements were taken for 3-4 days. The time series were analyzed with respect to the midline estimating statistic of rhythm (i.e., mean value of a periodic event after fit to a cosine function), amplitude, and acrophase (i.e., phase angle that corresponds to the peak of a given period) of the 24-h period. The LDF signal displayed a significant circadian rhythm, with the peak occurring at around midnight in SDR and TGR, despite inverse periodicity of ABP in TGR. This finding suggests independence of LDF periodicity from ABP regulation. Furthermore, the acrophase of the LDF was consistently found before the acrophase of the activity. From the present data, it is concluded that there is a circadian periodicity in the regulation of cerebral perfusion that is independent of circadian changes in ABP and probably is also independent of locomotor activity. The presence of a circadian periodicity in CBF may have implications for the occurrence of diurnal alterations in cerebrovascular events in humans.