Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.     

Compensatory sleep responses to wakefulness induced by the dopamine autoreceptor antagonist (-)DS121

The effect of the dopamine autoreceptor antagonist (-)DS121 on wakefulness, locomotor activity, body temperature and subsequent compensatory sleep responses was examined in the rat. Animals entrained to a light-dark cycle were treated at 5 h after lights-on (CT-5) with 0.5, 1, 5 or 10 mg/kg i.p. (-)DS121 or methylcellulose vehicle. An additional group received 5 mg/kg i.p. (-)DS121 or vehicle 6 h after lights-off (CT-18). At CT-5, (-)DS121 dose-dependently increased wakefulness, locomotor activity and body temperature, and decreased both non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) during the first 4 h post-treatment relative to vehicle controls. REM interference lasted up to 3 h longer than NREM. Low doses of (-)DS121 (0.5 and 1 mg/kg) produced relatively little waking that was not followed by significant compensatory sleep responses. In contrast, higher doses (5 and 10 mg/kg) produced compensatory hypersomnolence (robust increases in NREM immediately after the primary waking effect) that was proportional to the duration of drug-induced wakefulness. NREM recovery 24 h post-treatment was the same for the 5 mg/kg (65.4 +/- 9.9 min) and 10 mg/kg (64.8 +/- 9.3 min) doses, but was not proportional to prior wake duration. NREM displaced by drug-induced wakefulness was recovered completely by 24 h post-treatment at the 5 mg/kg dose, but only 63.5% recovered at 10 mg/kg. In contrast, equivalent wakefulness produced by sleep deprivation yielded 100% NREM recovery. At CT-18, (-)DS121 (5 mg/kg) increased wakefulness without disproportionately increasing locomotor activity, and was compensated fully by 24 h post-treatment. These data show that (-)DS121 dose-dependently increases wakefulness, which is followed by hypersomnolence that is proportional to drug-induced wake-promoting efficacy.     

Hemoglobins Aida (alpha 64 Asp leads to Asn) and D-Los Angeles (beta 121 Glu leads to Gln) in an Asian-Indian family

Hemoglobins D-Los Angeles (beta 121 Gln) and Aida (alpha 64 Asn) were encountered in an Asian Indian, unassociated with any clinical manifestations. These hemoglobins had normal oxygen affinities and were stable to heat and isopropanol. The ratio of alpha chain synthesis to beta chain synthesis was close to unity. The identical change in isoelectric points of the two variants produced an interesting electrophoretic pattern.     

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study

Increasing recognition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimer's disease (AD) were examined immunohistochemically with antibodies to domain-specific sites of APP (N-terminal, C-terminal, beta A4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with beta A4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed only rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to beta A4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled only cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.     

Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis)

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.     

Skin lesions in dogs, horses and calves caused by the stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Specific skin lesions caused by Stomoxys calcitrans on the feeding sites of different species are described. Skin lesions appeared on dogs, horses and calves following bites of stable flies. Necrotic dermatitis was observed in 32 dogs of various breeds at the tip of the ears. Exudative dermatitis appeared on the legs of 45 adult horses and dermatitis was diagnosed in the "hair whirlpools" on the backs of 18 white calves.     

Parkinson''s disease, amyotrophic lateral sclerosis and spinal muscular atrophy are caused by an unstable (CAG)n trinucleotide repeat microsatellite

The influence of previous trauma in the management of patients with temporomandibular disorders (TMD) is controversial. The objectives of this study were to compare treatment regimens and outcomes in motor vehicle accident trauma-related versus nontrauma-related TMD patients. Files of 50 trauma and 50 matched nontrauma TMD patients were reviewed. Information concerning treatment received, progress of symptoms with treatment, and findings from the final examination were recorded. As a whole group, posttraumatic TMD patients tended to receive more types of treatment (P < .0001), have more medications prescribed (including analgesics, P < .001; nonsteroidal anti-inflammatory drugs, P = .001; muscle relaxants, P = .001; and tricyclic antidepressants, P < .001), have more oral medicine clinic visits (P = .07) over a longer period of time (P = .06), and have a poorer treatment outcome (P < .001) as compared to the nontrauma group. When the patients were separated into TMD diagnostic classification subsets, only some of these differences between trauma and nontrauma patients were seen, but the subset group sizes were small and only a few of the groups could be compared. There did not seem to be a significant effect from settling insurance claims prior to the last clinic visit. Trauma may be an important prognostic factor in the management of some TMD patients.     

Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28

Actin-binding protein-280 (ABP-280) is a dimeric actin filament crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. We have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus.     

A comment on the recent study of the Mechanical Comprehension Test (CC) by R. L. Decker.

Reference is made to a 1959 article (see 33: 6982) which is "lacking in accuracy to such an extent that some comments on it appear to be in order." (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Phenotype variability of the dominant beta-thalassemia induced in four Dutch families by the rare cd121 (G-->T) mutation

BACKGROUND: Initial clinical experience with recombinant factor VIIa (rVIIa) for treatment of haemophilia patients with inhibitors against factor VIII or IX has been obtained by administration of rVIIa by repeated intravenous bolus injections. However, continuous infusion of rVIIa may be a more appropriate administration method if prolonged treatment is indicated. METHODS: We have surveyed and analysed the initial experience with continuous infusion of rVIIa in the Netherlands and Belgium. RESULTS: Five hospitals treated 7 haemophilia patients with inhibitors on 9 different occasions (4 bleedings, 5 surgical interventions) by continuous infusion of rVIIa over a total of 59 days. Haemostatic coverage was considered effective in 8 out of 9 cases and partially effective in 1 case. Continuous infusion of rVIIa was aimed at rVIIa target plasma levels of 10 U/ml and a decrease in prothrombin time (PT) of 3 s compared to control levels. This was obtained by an initial bolus injection of 90 micrograms/kg prior to continuous infusion of rVIIa at doses between 30-6 micrograms/kg/h (mean 17.5 micrograms/kg/h). A conventional one-stage factor VII coagulation assay, often used in combination with a PT, was satisfactory in monitoring rVIIa treatment. The additional clinical value of anti-fibrinolytic and anti-thrombophlebitic treatment was unclear. CONCLUSION: In our experience, rVIIa appeared to be efficacious and safe when administered by continuous infusion. Continuous infusion of rVIIa is more convenient than bolus injections or rVIIa, easy to monitor and provides a cost reduction of > 50%. These advantages make continuous infusion an attractive administration method for prolonged treatment with rVIIa.     

Defects in courtship and vision caused by amino acid substitutions in a putative RNA-binding protein encoded by the no-on-transient A (nonA) gene of Drosophila

The Drosophila no-on-transient A (nonA) gene is involved in the visual behaviors and courtship song of the fly. The NONA polypeptide contains two copies of the RNA-recognition motif (RRM), a hallmark of proteins involved in RNA binding, and an adjacent conserved charged region. This 311-amino-acid region is found in four other proteins and largely overlaps the Drosophila-Behavior/Human Splicing (or DBHS) domain. The newest family member, Drosophila nAhomo, was discovered in a database search, and encodes a protein with 80% identity to NONA. In this study, three nonA mutations generated by chemical mutagenesis were sequenced and found to fall within the conserved region. Site-directed mutagenesis of the two RRMs, and within a (conserved) charged region located C-terminal to them, was performed to determine the significance of these domains with respect to whole-organismal phenotypes. Behavior and viability were assessed in transformed flies, the genetic background of which lacks the nonA locus. Point mutations of amino acid 548 in the charged region confirmed the etiology of the nonAdiss courtship-song mutation and showed that a milder substitution at this site produced intermediate singing behavior, although it failed to rescue visual defects. Mutagenesis of the RRM1 domain resulted in effects on viability, vision, and courtship song. However, amino acid substitutions in RNP-II of RRM2 led to near-normal phenotypes, and the in vivo nonA mutations located in or near RRM2 caused visual defects only. Thus, we suggest that the first RRM could be important for all functions influenced by nonA, whereas the second RRM may be required primarily for normal vision.     

Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein

Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H-2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus.     

Swelling in the isolated perfused cornea induced by 12(R)hydroxyeicosatetraenoic acid

PURPOSE: To evaluate the effect of 12(R)hydroxyeicosatetraenoic acid (12(R)HETE) on corneal swelling when directly perfused to human and rabbit corneal endothelium. METHOD: Excised rabbit and human corneas were mounted in the in vitro specular microscope and the endothelium was perfused with 12(R)HETE at 10(-5), 10(-6), and 10(-7) mol/l. Both 12(R)HETE and 12(S)HETE were compared at equal molar (10(-6) mol/l) concentrations. The reversal of 12(R)HETE and ouabain corneal swelling was also compared. Endothelial permeability to carboxyfluorescein was measured after 12(R)HETE perfusion. High-performance liquid chromatographic analysis confirmed that 12(R)HETE remained in the perfusion media. RESULTS: 12(R)HETE caused a dose-dependent corneal swelling of 25 +/- 2, 24 +/- 1, and 14 +/- 0.5 microns/hr at 10(-5), 10(-6), and 10(-7) mol/l, respectively. Equal molar concentrations (10(-6) mol/l) of 12(S)HETE did not cause corneal swelling. Removal of the 12(R)HETE from the perfusion media resulted in reversal of corneal swelling whereas corneal swelling induced by ouabain did not reverse after ouabain removal. 12(R)HETE (10(-6) mol/l) perfused to the human corneal endothelium inhibited temperature reversal corneal thinning when compared to the paired corneal endothelium perfused with BSS Plus (Alcon Laboratories, Inc., Fort Worth, TX). Na/K adenosine triphosphatase activity was inhibited by 10(-6) mol/l ouabain by 35%, 10(-6) mol/l 12(R)HETE by 54%, and 10(-6) mol/l 12(S)HETE by 0.5%. Endothelial permeability to carboxyfluorescein was unaffected by 12(R)HETE. CONCLUSION: 12(R)HETE causes corneal swelling by inhibiting endothelial pump function. This inhibition of transport appears to be at least partly mediated by inhibition of endothelial Na/K adenosine triphosphatase.     

Deletion of an endosomal/lysosomal targeting signal promotes the secretion of Alzheimer's disease amyloid precursor protein (APP)

Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.     

Serum Clara cell protein (CC16): a new valid marker of the distal airway damages caused by air pollutants in rats

Two brothers, 17 and 21 years of age, with depressed nasal bridge, prominent frontal bones, hypoplastic maxilla, mild sensorineural hearing loss, broad terminal phalanges and mild pulmonary stenosis are presented. These findings are similar to those of the syndrome described by Keipert et al. in 1973. To the best of our knowledge, this is only the second report of this syndrome.     

Alterations in protein aggregation and degradation due to mild and severe missense mutations (A104D, R157N) in the human phenylalanine hydroxylase gene (PAH)

Although the importance of the father for the development of the child is well known and although an increased presence of the father in the family system is often demanded, processes of active shutting out the father occur. The deterioration of relation between father and child is not seen as a result of pathological male personality, but rather as a result of family or couple system. The special situation for counsellors of such cases is discussed.     

Fucosterol-24,28-epoxide, an intermediate in the oxidative biodegredation of fucosterol to cholesterol by the cricket (Locusta migratoria (R. and F.)

beta-Sitosterol 1 is metabolised to cholesterol 5 by phytophagous insects. It has been previously shown that fucosterol-24,28 epoxide 3 is transformed into 5 in Locusta migratoria, desmosterol 4 being an intermediate. It is now established that locusts transform [3-3H]fucosterol propionate into the corresponding labelled epoxide 3, recovered as such or as an oxazoline derivative 11.