Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

Mutation analysis of feline Niemann-Pick C1 disease

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.     

A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

Hepatitis E virus (HEV) is the causative agent of hepatitis E, anacute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2protein assembled as dimers, trimers and higher order forms. WhileN-terminal deletions up to 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.     

Effects of heparin on platelet aggregation and release and thromboxane A2 production.

Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with 3H-arachidonic acid and 14C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of 14C-serotonin and 3H-arachidonic acid metabolites, it appeared that either release of 14C and 3H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both 14C and 3H.     

Association between the DRD2 A1 allele and opium addiction in the Iranian population.

Dysfunction of the central dopaminergic neurotransmission has been suggested to play an important role in the etiology of certain neuropsychiatric disorders such as drug abuse. It has been shown that the dopamine D2 receptor (DRD2) gene dysfunction is associated with multi-drug addiction. Addiction to opium is the most common form of drug abuse in Iran. We studied the allelic association between DRD2 Taq I A polymorphism in 100 opium-dependent Iranian patients and 130 unrelated controls. A 310 bp (base pair) region surrounding Taq I site at the DRD2 locus was amplified by polymerase chain reaction (PCR) and the PCR product was incubated with Taq I restriction enzyme. The A1 allele remained intact while the A2 allele was cut. Significant association was observed between A1 allele and addiction in the patients group (P < 0.0001). Moreover, the frequency of A1A1 genotype was significantly higher in opium users than controls (P < 0.0001). Our result indicates that DRD2 might be involved in the pathophysiology of opium addiction.     

Two new species of Eimeria Schneider 1875 (Apicomplexa: Eimeriidae) from the broad-toothed field mouse, Apodemus mystacinus Danford and Alston 1877 (Rodentia: Muridae) from Jordan

Coprological examination of 40 Apodemus mystacinus Danford and Alston 1877 from Jordan revealed oocysts of three species of genus Eimeria. Two species are described as new. Eimeria zuhairamri sp. n. has broadly ellipsoidal oocysts 29.6 (27.0–34.0)×23.3 (22.0–25.0) m with distinctly granulated wall and oocyst residuum. Endogenous development occurs in jejunum and ileum. Eimeria alorani sp. n. has oocysts 26.9 (23.0–29.0)×19.3 (18.0–22.0) m with smooth wall and absent residuum. Endogenous development is confined to the caecum. The third species, developing in jejunum, has oocysts morphologically indistinguishable from Eimeria uptoni. The identity of E. uptoni and the taxonomy of Eimeria of Apodemus are discussed.     

GJB2 mutations: passage through Iran

Hereditary hearing loss (HHL) is a very common disorder. When inherited in an autosomal recessive manner, it typically presents as an isolated finding. Interestingly and unexpectedly, in spite of extreme heterogeneity, mutations in one gene, GJB2, are the most common cause of congenital severe-to-profound deafness in many different populations. In this study, we assessed the contributions made by GJB2 mutations and chromosome 13 g.1777179_2085947del (the deletion more commonly known as del (GJB6-D13S1830) that includes a portion of GJB6 and is hereafter called Delta(GJB6-D13S1830)) to the autosomal recessive non-syndromic deafness (ARNSD) genetic load in Iran. Probands from 664 different nuclear families were investigated. GJB2-related deafness was found in 111 families (16.7%). The carrier frequency of the 35delG mutation showed a geographic variation that is supported by studies in neighboring countries. Delta(GJB6-D13S1830) was not found. Our prevalence data for GJB2-related deafness reveal a geographic pattern that mirrors the south-to-north European gradient and supports a founder effect in southeastern Europe.     

Hyperbaric oxygenation mitigates focal cerebral injury and reduces striatal dopamine release in a rat model of transient middle cerebral artery occlusion

The usefulness of the administration of hyperbaric oxygen (HBO) in the treatment of acute focal cerebral ischemia remains debatable. A significant association exists between focal cerebral injury and an excessive release of extracellular dopamine (DA). In vivo microdialysis was used in the present study to examine the effect of HBO on DA release in the striatum during ischemia and reperfusion in rats. The histological changes occurring were also evaluated. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCA) using a surgically placed intraluminal filament. Control rats (n=8) were subjected to 1 h of ischemia, whilst the study rats (n=8) were in addition treated with HBO (2.8 atmospheres of absolute pressure 100% O₂) during ischemia. Both groups were returned to breathing room air at normal pressure during reperfusion. Microdialysis samples were continuously collected at 15 min intervals at 2 μl·min^–1. The [mean (SE)] increase in release of striatal DA attained significance after 30 min of occlusion of MCA [170 (24)%], and continued to increase [268 (26)% at 45 min] reaching a peak level at 60 min [672 (59)%] before returning to the baseline level during the late reperfusion phase. There was no significant change in the level of DA in HBO treated rats during the period of ischemia. A significant reduction in edema and neuronal shrinkage were observed by histological examination in HBO treated rats when compared to the control rats. The results showed that HBO, when administered during ischemia, offered significant neuroprotection in our experimental model of transient focal cerebral ischemia in the rat. The mechanism seems to imply, at least in part, a reduced level of DA. Electronic Publication     

Bryostatin 1-induced modulation of nucleoside transporters and 2-chlorodeoxyadenosine influx in WSU-CLL cells

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.