Recombinant glutathione S-transferase/CD36 fusion proteins define an oxidized low density lipoprotein-binding domain

CD36 is a multifunctional cell-surface receptor that binds adhesion molecules such as thrombospondin-1 and collagen and modified lipids and/or lipoproteins. It participates in cellular uptake of photoreceptor outer segments and scavenging of apoptotic cells and oxidized low density lipoprotein (Ox-LDL). Recognition and internalization of Ox-LDL by mononuclear phagocytes may play an important role in the development of atherosclerotic lesions. We have utilized a series of recombinant bacterial glutathione S-transferase/CD36 fusion proteins that span nearly all of the CD36 molecule to characterize the structural domain on CD36 that recognizes Ox-LDL. We found that the Ox-LDL-binding domain is different from the thrombospondin-1-binding domain located at amino acids 93-120. A fusion protein containing the region extending from amino acids 5 to 143 formed specific, saturable, and reversible complexes with Ox-LDL. As with intact CD36, binding was blocked by excess unlabeled Ox-LDL and antibodies to CD36. The stoichiometry and affinity of the fusion protein for Ox-LDL were similar to those of the intact protein. We also demonstrated that this fusion protein competitively inhibited binding of Ox-LDL to purified platelet CD36 and to CD36 expressed on peripheral blood monocytes and CD36 cDNA-transfected melanoma cells. The use of smaller peptides and fusion proteins including those spanning amino acids 28-93 and 5-93 has further narrowed the binding site to a region from amino acids 28 to 93, although participation of a sequence in the noncontiguous region 120-155 cannot be excluded. This study, for the first time, demonstrates unique regions of the scavenger receptor CD36 that bind the Ox-LDL ligand. Our structural analysis of the receptor provides information as to potential control of the trafficking of modified lipoproteins into the blood vessel wall.     

Clomiphene analogs with activity in vitro and in vivo against human breast cancer cells

The masking procedure by Paradiso and Nakayama (1991) (Vision Research, 31, 1221-1236) was used to investigate brightness filling-in within textures made of line elements: a texture stimulus was masked by a second stimulus containing a square contour. When a uniform texture was presented, the texture region inside the masking square appeared darkened and a small number of texture elements were perceived with a degenerated shape, appearing as dim dots or shorter line elements; it is as if the line element expanded from a bright point to fill the entire region defined by its contour. If the texture stimulus was a texture patch segregating from the surrounding texture by an orientation gradient and this patch was inside the square mask, darkening was not as strong as in the previous condition, and masked line elements preserved their elongated shape. Brightness spreading was measured in two experiments using dichoptic presentations. Experiment 1 used an adjustment task and showed that the brightness of texture line elements spread from equiluminant borders between segregating textures. Experiment 2 used a matching task and demonstrated that spreading was blocked by segregation borders dependent on the orientation gradient between texture line elements. The selectivity for line orientation began 40-80 msec after texture onset and maximal spreading occurred at approximately 120 msec. These findings may indicate that two processes subserve filling-in within textures: the first spreads isotropically the mean stimulus luminance at an initial processing stage of image analysis; at a later stage, the second spreads a texture flow (both brightness and shape of line elements) directed along the orientation of texture line elements. The texture flow mechanism fills in with a texture surface the region bounded by segregation contours.     

Angiotensin-converting enzyme inhibition delays pulmonary vascular neointimal formation

Primary pulmonary hypertension (PPH) is a disease characterized pathologically by pulmonary artery medial hypertrophy, adventitial thickening, and neointimal proliferation. Increasing recognition of the importance of remodeling to the pathogenesis of PPH suggests new therapeutic possibilities, but it will be necessary to (1) identify essential mediators of remodeling, and (2) demonstrate that inhibiting those mediators suppresses remodeling before new antiremodeling therapies can be considered feasible. The effect of angiotensin-converting enzyme (ACE) inhibition on pulmonary vascular remodeling was studied in a newly developed rat model in which neointimal lesions develop between 3 and 5 wk after monocrotaline injury is coupled with increased pulmonary artery blood flow after contralateral pneumonectomy. Neointimal formation was significantly suppressed at 5 wk by ACE inhibition whether it was started 10 d before or 3 wk after remodeling was initiated, although medial hypertrophy and adventitial thickening still developed. By 11 wk, the extent of neointimal formation in rats treated with ACE inhibition was similar to rats without ACE inhibition at 5 wk. Pulmonary artery pressures and right ventricular weights correlated with the extent of neointimal formation. Northern blot analysis and in situ hybridization demonstrated marked suppression of lung tropoelastin and type I procollagen gene expression in the presence of ACE inhibition. An angiotensin II type I receptor antagonist partially, but not completely, replicated the effects of ACE inhibition. These data suggest that the tissue angiotensin system may be a target for therapeutic efforts to suppress the vascular remodeling that is characteristic of primary pulmonary hypertension.     

Dideoxy fingerprinting: application to the genotyping of Echinococcus

Dideoxy fingerprinting is an efficient method for the detection of sequence variation in PCR-amplified DNA segments. It is a hybrid between single-strand conformation polymorphism and dideoxy sequencing, employing only one dideoxynucleotide in the sequencing reaction. Herein, we report the application of dideoxy fingerprinting to genetically type cestodes of the genus Echinococcus, utilising the mitochondrial cytochrome c oxidase subunit I as the gene sequence for analysis. All of the seven genotypes (G1, G4, G6, G8, O, V and M2) examined could be readily differentiated from one another by their characteristic and reproducible dideoxy fingerprinting profiles. Only subtle variation in profiles was detected among some of the eight isolates representing genotype G1, and no variation was detected between two samples of genotype G4 and of genotype M2. The capacity of dideoxy fingerprinting to detect all nucleotide variations over 150-250bp fragments indicates that it should be possible to distinguish among all of the genotypes of Echinococcus thus far described. Although employed herein to display sequence variation in the cytochrome c oxidase subunit I of Echinococcus, dideoxy fingerprinting could be used for the high-resolution analysis of nucleotide variations in other parasite genes, without the need for DNA sequencing. This has important implications for studying the genetic structure of parasite populations.     

The use of transitional implants for immediate fixed temporary prostheses in cases of implant restorations

While the original Br?nemark implant protocol has continued to evolve, the avoidance of implant loading during osseointegration remains a prerequisite with all implant systems. Immediately loaded transitional implants have recently been developed to support the fabrication of a fixed provisional prosthesis that provides implant patients with improved aesthetics and function during the osseointegration period. In this manner, osseointegration can occur free from prosthetic and transmucosal loads. This article describes the use of transitional implants and presents a classification of three different case types.     

Low Thermal Budget Processing for Sequential 3-D IC Fabrication

Laser annealing can be used for electrical activation of dopants without excessively heating the material deeper within the work piece. The authors demonstrate that laser annealing could be used for activating the dopants in the upper levels of an exemplary 3-D integrated circuit structure without affecting the operation of the devices below. We then use a 450 degC low-temperature oxide deposition process for forming the gate oxide and laser annealing for activating the dopants at the source/drain and gate regions to fabricate CMOS transistors. This process can be used to fabricate the transistors on the upper levels of a general 3-D IC structure without affecting the quality of the devices below     

Neurorehabilitation outcome in moyamoya disease

OBJECTIVE: To assess the contribution of emotional health problems to the burden of disability affecting people of working age. DESIGN: Analysis of data collected in a postal questionnaire survey of a random sample of people aged 18-64 years. SETTING: The four counties of the old Oxford region in 1991. SUBJECTS: 9332 people who responded to a questionnaire survey mailed to 14,000 people randomly selected from the Family Health Service Authority registers of the four counties of Berkshire, Buckinghamshire, Oxfordshire, and Northamptonshire. OUTCOMES MEASURES: Interference with work or other regular daily activity as reported in questions 4 and 5 of the health status measure SF-36. RESULTS: In this population the prevalence of disability attributable to emotional health problems was greater than that attributable to all physical health problems combined. People reporting that their work or other regular daily activity was affected by their emotional health were much less likely to report a long-standing illness, consultation with a GP or consultation with a hospital doctor than people reporting a physical health problem. CONCLUSIONS: Emotional health problems are a more important cause of disability in adults of working age than all physical health problems put together. Their importance is underestimated in health needs assessment exercises, which are based on NHS consultation rates or reporting of chronic illness. Research into the causes, prevention, and management of emotional health problems should be a national priority for the health service.     

Evaluation of plasma natriuretic peptides as markers for left ventricular dysfunction

To test the hypothesis that elevated plasma levels of natriuretic peptides may serve to identify patients with left ventricular (LV) dysfunction, we assessed the predictive diagnostic value of natriuretic peptide levels, in addition to clinical and electro-cardiographic risk factors, as noninvasive indicators of cardiac dysfunction. Plasma levels of atrial natriuretic peptide (cANP) (99-126), N-terminal fragment of proANP (nANP) (26-55), nANP(80-96), brain natriuretic peptide (BNP-32), proBNP(22-46), and C-type natriuretic peptide (CNP-22) were measured in 211 subjects before cardiac catheterization. The strongest correlations with parameters of LV function were found for nANP(80-96) (up to r = -0.55, p < 0.0001), whereas there was no significant correlation with proBNP(22-46) or CNP-22. In patients with LV ejection fractions (LVEF) < or = 45% (n = 38) nANP(26-55), nANP(80-96), cANP(99-126), and BNP-32 were significantly increased (p < 0.001). Partition values for elevated versus normal natriuretic peptide levels were obtained from normal controls and used to separate subjects with and without LV dysfunction. Receiver operating characteristic analysis for LVEF < or = 45% indicated a significantly better diagnostic accuracy for high levels of nANP(80-96), nANP(22-56), cANP(99-126), and BNP-32 than for proBNP and CNP-22. Multivariate analysis by logistic regression identified Q waves and bundle branch block in the electrocardiogram as well as elevated plasma levels of cANP, nANP(80-96), and nANP(26-55) as the strongest independent predictors of low ejection fractions. The relative risk of LV dysfunction was raised up to tenfold in subjects with high natriuretic peptide levels (p < 0.001). The addition of nANP(80-96) and nANP(26-55) to the combination of clinical and electrocardiographic risk factors did not further improve the diagnostic sensitivity for the detection of LVEF < or = 45%, but it markedly increased the overall accuracy (59% to 81%, p < 0.001) and specificity (55% to 81%, p < 0.001). Among natriuretic peptides, elevated nANP(80-96) and nANP(26-55) levels have the strongest impact on the detection of LV dysfunction. They add to the diagnostic information contained in clinical and electrocardiographic factors. Plasma levels alone or in combination with clinical factors seem to be of value for a refined identification of abnormal LV function in the individual patient.     

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.     

FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development

The development of calvarial bones is tightly co-ordinated with the growth of the brain and needs harmonious interactions between different tissues within the calvarial sutures. Premature fusion of cranial sutures, known as craniosynostosis, presumably involves disturbance of these interactions. Mutations in the homeobox gene Msx2 as well as the FGF receptors cause human craniosynostosis syndromes. Our histological analysis of mouse calvarial development demonstrated morphological differences in the sagittal suture between embryonic and postnatal stages. In vitro culture of mouse calvaria showed that embryonic, but not postnatal, dura mater regulated suture patency. We next analysed by in situ hybridisation the expression of several genes, which are known to act in conserved signalling pathways, in the sagittal suture during embryonic (E15-E18) and postnatal stages (P1-P6). Msx1 and Msx2 were expressed in the sutural mesenchyme and the dura mater. FGFR2(BEK), as well as Bmp2 and Bmp4, were intensely expressed in the osteogenic fronts and Bmp4 also in the mesenchyme of the sagittal suture and in the dura mater. Fgf9 was expressed throughout the calvarial mesenchyme, the dura mater, the developing bones and the overlying skin, but Fgf4 was not detected in these tissues. Interestingly, Shh and Ptc started to be expressed in patched pattern along the osteogenic fronts at the end of embryonic development and, at this time, the expression of Bmp4 and sequentially those of Msx2 and Bmp2 were reduced, and they also acquired patched expression patterns. The expression of Msx2 in the dura mater disappeared after birth. FGF and BMP signalling pathways were further examined in vitro, in E15 mouse calvarial explants. Interestingly, beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts, but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume both when placed on the osteogenic fronts and on the mid-sutural area, but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. We suggest that the local application of FGF on the osteogenic fronts accelerating suture closure in vitro, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors. Taken together, our data suggest that conserved signalling pathways regulate tissue interactions during suture morphogenesis and intramembranous bone formation of the calvaria and that morphogenesis of mouse sagittal suture is controlled by different molecular mechanisms during the embryonic and postnatal stages. Signals from the dura mater may regulate the maintenance of sutural patency prenatally, whereas signals in the osteogenic fronts dominate after birth.