Measurement of Cosmic Ray and Trapped Proton LET Spectra on the STS-95 HOST Mission.

This paper reports on in situ measurements of the Linear-Energy-Transfer (LET) spectra of galactic cosmic rays and their progeny and of trapped Van Allen belt protons as recorded by a Pulse Height Analyzer (PHA) radiation spectrometer which flew on the STS-95 DISCOVERY mission on the Hubble Orbital Systems Test (HOST) cradle. The Shuttle was launched on 29 October 1998 and had a mission duration of 8.5 days during the minimum phase of the solar activity cycle. The orbit of the STS-95 was about 550 km altitude and 28.5° inclination. Close agreement was seen between radiation environment model predictions and the measurements of the PHA. Agreement is obtained by considering the directionality of the radiation interacting with the Shuttle structure.

Microarray analysis of normal and abnormal chick ventricular myocardial development.

The left and right ventricle originate from distinct parts of the cardiac tube, and several genes are known to be differentially expressed in these compartments. The aims of this study were to determine developmental differences in gene expression between the left and right ventricle, and to assess the effect of altered hemodynamic loading. RNA was extracted from isolated left and right normal chick embryonic ventricles at embryonic day 6, 8, and 10, and from day 8 left atrial ligated hearts with hypoplastic left and dilated right ventricles. cRNA was hybridized to Affymetrix Chicken Genome array according to manufacturer protocols. Microarray analysis identified 302 transcripts that were differentially expressed between the left and right ventricle. Comparative analysis detected 91 genes that were different in left ventricles of ligated hearts compared to age-matched ventricles, while 66 were different in the right ones. A large number of the changes could be interpreted as a delay of normal maturation. The approach described in this study could be used as one of the measures to gauge success of surgical procedures for congenital heart disease and help in determining the optimal time frame for intervention to prevent onset of irreversible changes.

Role of subchondral bone during early-stage experimental TMJ osteoarthritis.

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease that affects both cartilage and subchondral bone. We used microarray to identify changes in gene expression levels in the TMJ during early stages of the disease, using an established TMJ OA genetic mouse model deficient in 2 extracellular matrix proteins, biglycan and fibromodulin (bgn(-/0)fmod(-/-)). Differential gene expression analysis was performed with RNA extracted from 3-week-old WT and bgn(-/0)fmod(-/-) TMJs with an intact cartilage/subchondral bone interface. In total, 22 genes were differentially expressed in bgn(-/0)fmod(-/-) TMJs, including 5 genes involved in osteoclast activity/differentiation. The number of TRAP-positive cells were three-fold higher in bgn(-/0)fmod(-/-) TMJs than in WT. Quantitative RT-PCR showed up-regulation of RANKL and OPG, with a 128% increase in RANKL/OPG ratio in bgn(-/0)fmod(-/-) TMJs. Histology and immunohistochemistry revealed tissue disorganization and reduced type I collagen in bgn(-/0)fmod(-/-) TMJ subchondral bone. Early changes in gene expression and tissue defects in young bgn(-/0)fmod(-/-) TMJ subchondral bone are likely attributed to increased osteoclast activity. Analysis of these data shows that biglycan and fibromodulin are critical for TMJ subchondral bone integrity and reveal a potential role for TMJ subchondral bone turnover during the initial early stages of TMJ OA disease in this model.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes.

AIMS/HYPOTHESIS: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. METHODS: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. RESULTS: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. CONCLUSIONS/INTERPRETATION: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy.

Cubilin and megalin: partners in lipoprotein and vitamin metabolism.

Members of the low-density lipoprotein receptor (LDLR) family are unrivalled for their ability to endocytose and target ligands to lysosomes for degradation. Their endocytic and catabolic functions make them essential to homeostatic regulation of the level and activity of their ligands in biological fluids and interstitial spaces. Over the last few years it has become evident that the endocytic function of members of the LDLR family is employed by other kinds of cell surface receptors. Recently, the low-density lipoprotein receptor related protein-2 (megalin) was shown to act in concert with cubilin, a receptor for high-density lipoproteins (HDL)/apolipoprotein A-I (apoA-I), intrinsic factor-vitamin B12 and albumin to mediate ligand endocytosis. In this article, we review the state of knowledge pertaining to cubilin and megalin, emphasizing their joint roles in both lipoprotein and vitamin metabolism.

Unrecognized benign paroxysmal positional vertigo in elderly patients.

Balance disorders in elderly patients are associated with an increased risk of falls but are often difficult to diagnose because of comorbid chronic medical problems. We performed a cross-sectional study to determine the prevalence of unrecognized benign paroxysmal positional vertigo (BPPV) and associated lifestyle sequelae in a public, inner-city geriatric population. Dizziness was found in 61% of patients, whereas balance disorders were found in 77% of patients. Nine percent were found to have unrecognized BPPV. Multivariate analysis demonstrated that the presence of a spinning sensation and the absence of a lightheadedness sensation predicted the presence of unrecognized BPPV. Patients with unrecognized BPPV were more likely to have reduced activities of daily living scores, to have sustained a fall in the previous 3 months, and to have depression. These data indicate that unrecognized BPPV is common within the elderly population and has associated morbidity. Further prospective studies are warranted.

Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins.

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

An Oct-like binding factor regulates Myf-5 expression in primary avian cells.

Myogenic regulatory factors (MRFs) are hierarchical regulators of skeletal myogenesis. Many MRF promoters have been well characterized with respect to flanking sequences that control their expression. Yet the promoter elements that regulate Myf-5, the first MRF expressed during mammalian embryogenesis, are still largely unknown. Comparison of Myf-5 5' flanking regions from bovine, mouse, and chicken genes revealed three evolutionarily conserved elements proximal to the transcription start site: the TATA box, an octamer motif, termed OLS, and a 6-bp C-rich element. Mobility shift assays and DNase I footprinting analysis demonstrated that a nuclear factor(s) present in both bovine and avian muscle and nonmuscle tissues specifically recognized OLS. Furthermore, this binding activity reacted with a polyclonal Oct-1 antibody. In avian primary myoblast and fibroblast cultures, CAT reporter constructs under regulation of the proximal Myf-5 5' flanking sequence were expressed preferentially in myoblasts with CAT levels approximately 12-fold higher than in fibroblasts. The TATA box and octamer motif were important for expression in both myoblasts and fibroblasts: loss of the TATA box abolished activity, and disruption of the OLS resulted in 50-75% loss of promoter activity.

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene.

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.

The low earth orbit environment observed using CREAM and CREDO.

The Cosmic Radiation Environment and Dosimetry experiment (CREDO) has been operational on board the Advanced Photovoltaics & Electronics Experiment Spacecraft since August 1994. Extensive measurements of cosmic ray linear energy transfer spectra (using data to January 1996) and total dose (using data to November 1994) have been made, and compared with predictions of standard models. Detailed consideration of spacecraft shielding effects have been made. Predictions are shown to overestimate the measured linear energy transfer spectra. The CREAM experiment was flown on STS-63 in the SpaceHab module. Results show penetration of high energy electrons into the SpaceHab module.

Dexamethasone-mediated induction of MMTV-myf5 in DD3 myoblasts increases endogenous myogenin expression but does not transactivate myf5.

DD3 cells are a myoblast line generated by stable transfection of C3H10T1/2 fibroblasts with the bovine myf5 cDNA (bmyf) fused to the dexamethasone-inducible MMTV promoter. After treating proliferating cells with dexamethasone, bmyf transcripts were induced approximately fivefold within 1.5-2.5 h. Induction of bmyf was followed 4 h later by a similar increase in myogenin transcripts which was dependent on protein synthesis. The elevated level of myogenin transcripts in dividing cells was comparable to that observed in DD3 myoblasts after differentiation. The 4-h lag before the myogenin response suggested involvement of an intermediate factor. However, one possible factor, myocyte-specific enhancer binding factor (MEF-2) (a known enhancer of myogenin promoter activity) was neither detectable nor inducible by dexamethasone in proliferating cells. myoD transcripts were barely detectable and uninducible in proliferating cells but strongly upregulated during differentiation. There was also a transient twofold increase in mrf4 transcripts by dexamethasone treatment in dividing cells, while no changes were detected in the levels of Id, E12, or TnC messages. The mouse myf5 gene was silent and uninducible in DD3 cells under proliferating and differentiating conditions. We conclude that ectopic expression of MMTV-bmyf led to activation of three of the endogenous myogenic factor genes of which only myogenin showed a rapid and sustained response to dexamethasone-mediated induction of bmyf in dividing cells.

Isolation, sequence, and characterization of the bovine myogenic factor-encoding gene myf-5.

We report here the isolation, sequencing, and characterization of the bovine homologue of the myogenic factor-encoding gene myf-5 (termed bmyf). The transcription unit of the gene is 4.2 kb in length and contains two introns. Primer extension experiments mapped the transcription start point at 158 bp upstream from the predicted start codon and 29 bp downstream from a canonical TATA box. The 3' untranslated region contains four AATAAA sites, three of which are used to produce transcripts of 1.5, 2, and 3 kb in bovine fetal muscle RNA.

Risk versus effort in the assessment of motor fatigue.

The choice of probability leffort (COPE) devices require fatigued subjects to choose between risk and effort. In a first experiment, where fatigue was induced by an intense or by a prolonged motor task and where the required effort was perceptual, the fatigue did not generalize to the test mode. A second experiment used motor fatigue of the arm or leg, and tested with arm muscle effort, showing that subjects fatigued with either limb chose riskier alternatives in order to avoid the effort. The data correlated with self-rated fatigue, and closely paralleled earlier work using perceptual fatigue and perceptual effort.