Post-translational modification and intracellular localization of a splice product of CD46 cloned from human testis: role of the intracellular domains in O-glycosylation

We obtained a unique CD46 cDNA, STc/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CY). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum. This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42,000, approximately 20,000 smaller than conventional CD46. These properties of the STc/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STc/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types. Since the STc/CY4 isoform may behave like sperm CD46 in cellular localization and post-translational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STc/CY4. Rosette formation was seen most effectively between hamster eggs and STc/CY4-expressing CHO cells. These results infer that O-glycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule. The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.     

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.     

Concentrations of vascular endothelial growth factor in the sera of normal controls and cancer patients

Vascular endothelial growth factor (VEGF) is known to play crucial roles in tumor angiogenesis. We have investigated the circulating level of VEGF in sera from cancer patients as well as from healthy normal controls using a sensitive enzymatic immunoassay. Immunoreactive VEGF proteins were detectable in normal sera, and the cutoff level was determined to be 180 pg/ml. In examined patients with all types of cancer, including breast, gastrointestinal, hepatobiliary, and lung cancer, an aberrant increase in the circulating level of VEGF was detected. For example, in 137 primary breast cancer patients, 12 (8.8%) showed an aberrant increase in VEGF levels. This aberrant expression of VEGF in sera was significantly associated with the progression of the disease, and with VEGF protein expression in tumor tissues. In addition, a Western blot analysis confirmed the presence of the VEGF165 form in sera from a patient with recurrent breast cancer. It was concluded that VEGF was detectable in normal sera, and its level was increased in some populations of cancer patients. A positive angiogenesis regulator, VEGF might function as an endocrine growth factor, particularly for solid tumors.     

Buspirone enhances immobility in the forced swim test in mice

We studied the effects of buspirone and 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on duration of immobility in mice in the forced swim test. Buspirone [3-10 mg/kg, intraperitoneally (IP)] potently and dose dependently increased the duration of immobility in mice. In contrast, following a single dose of 8-OH-DPAT (1-3 mg/kg, IP), there was a dose-dependent decrease in the duration of immobility. Pretreatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (200 mg/kg, IP, 3 days before further drug treatment) did not alter the effects of buspirone or 8-OH-DPAT. The increase in the duration of immobility induced by buspirone (3 mg/kg, IP) was blocked by NAN-190 [1-(2-methoxyphenyl)-4-(4-[2-phthalimido]butyl)-piperazine hydrobromide, 1 mg/kg, IP], a postsynaptic 5-HT1A receptor antagonist. However, the effect of 8-OH-DPAT (1 mg/kg, IP) was not blocked by NAN-190 (1 mg/kg, IP). The effect of buspirone (3 mg/kg, IP) was blocked by apomorphine (0.3 mg/kg, IP), a dopamine receptor agonist. Based on the results of this study, it is suggested that the effects of buspirone and of 8-OH-DPAT on immobility in the forced swim test may occur through different mechanisms.     

Hemorrhagic and nonhemorrhagic stroke: diagnosis with diffusion-weighted and T2-weighted echo-planar MR imaging

PURPOSE: To determine if diffusion- and T2-weighted echo-planar magnetic resonance (MR) imaging can be used to detect acute hemorrhagic stroke and to differentiate hemorrhagic from nonhemorrhagic stroke. MATERIALS AND METHODS: A total of 118 examinations (diffusion- and T2-weighted MR imaging) in 19 patients with 27 nonhemorrhagic strokes and in six patients with seven hemorrhagic strokes were performed. The ratios of apparent diffusion coefficient and of signal intensity on T2-weighted MR images in lesions to those in contralateral control areas were calculated. RESULTS: Decreased ADC was shown in lesions of acute (0-3 days) hemorrhagic stroke, as well as in lesions of acute nonhemorrhagic stroke. Hypointense areas were seen on T2-weighted MR images in patients with acute hemorrhagic stroke, in contrast to normal to increased signal intensity in those with acute nonhemorrhagic stroke. Apparent diffusion coefficient tended to remain decreased in hemorrhagic stroke lesions even 100 days after onset, in contrast to the increased coefficient in nonhemorrhagic stroke lesions at the late chronic stage (31 days or older). CONCLUSION: Diffusion- and T2-weighted echo-planar MR imaging can be used to detect and distinguish between acute hemorrhagic and nonhemorrhagic stroke.     

Elevated plasma adrenomedullin level in hyperthyroidism

Adrenomedullin is a recently discovered peptide that was first purified from phaeochromocytoma tissue and has marked vasodilatory activity, causing hypotension. In thyrotoxicosis, various haemodynamic changes are observed, including an increase in cardiac output and heart rate with a concomitant decrease in peripheral vascular resistance. To evaluate the mechanism underlying these haemodynamic changes in thyrotoxicosis, we measured the plasma adrenomedullin concentration in thyrotoxic patients with Graves' disease. The plasma concentration of adrenomedullin was elevated in hyperthyroid patients (14.7 +/- 5.7 pmol L-1) compared with euthyroid control subjects (5.6 +/- 1.3 pmol L-1) (P < 0.001). The correlation between the plasma adrenomedullin concentration and serum free thyroid hormone levels was marginally significant. The mean blood pressure was relatively low in the face of an elevated plasma adrenomedullin level. Adrenomedullin may therefore be responsible for the vasodilatation observed in thyrotoxicosis.     

The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract

Vascularization of organs generally occurs by remodelling of the preexisting vascular system during their differentiation and growth to enable them to perform their specific functions during development. The molecules required by early vascular systems, many of which are receptor tyrosine kinases and their ligands, have been defined by analysis of mutant mice. As most of these mice die during early gestation before many of their organs have developed, the molecules responsible for vascularization during organogenesis have not been identified. The cell-surface receptor CXCR4 is a seven-transmembrane-spanning, G-protein-coupled receptor for the CXC chemokine PBSF/SDF-1 (for pre-B-cell growth-stimulating factor/stromal-cell-derived factor), which is responsible for B-cell lymphopoiesis, bone-marrow myelopoiesis and cardiac ventricular septum formation. CXCR4 also functions as a co-receptor for T-cell-line tropic human immunodeficiency virus HIV-1. Here we report that CXCR4 is expressed in developing vascular endothelial cells, and that mice lacking CXCR4 or PBSF/SDF-1 have defective formation of the large vessels supplying the gastrointestinal tract. In addition, mice lacking CXCR4 die in utero and are defective in vascular development, haematopoiesis and cardiogenesis, like mice lacking PBSF/SDF-1, indicating that CXCR4 is a primary physiological receptor for PBSF/SDF-1. We conclude that PBSF/SDF-1 and CXCR4 define a new signalling system for organ vascularization.     

Boron carbide coating by electromagnetically accelerated plasma spraying

A new system of electromagnetically accelerated plasma spraying (EMAPS) consisting of a pulsed high-current arc-plasma gun and a large flow rate pulsed powder injector has been developed to synthesize a hard and dense coating of boron carbide (B₄C) with a high adhesion. The plasma gun with a co-axial cylindrical electrode configuration generates electromagnetically accelerated arc plasma with a typical velocity and maximum pressure of 1.5–3.0 km/s and 1 MPa, respectively, by discharging a pulsed high current of about 100 kA in peak and about 300 μsec of duration. The heating and accelerating of source powder are accomplished by injecting it into the inter-electrode space of the gun prior to the plasma generation using a newly developed pulsed powder injector that enables a gram of powder to be injected within 1 ms with precisely controlled time delay. With this system, hard B₄C coatings with a high adhesion and crystallinity were successfully formed on mirror-polished stainless (SUS304) substrates without a binder.     

Nonstationary peeling apparatus

A piece of apparatus for measuring the peel adhesion behavior of pressure-sensitive adhesive tapes was developed. The apparatus allows peel rates to be varied as any functions of time or peel distance. In a cycle run of the accelerating and decelerating peel processes, nonstationary peeling was investigated over a wide range of peel rates using relatively short-length samples. The resultant behavior in nonstationary peeling indicated that in the interfacial failure regime, a good agreement with the normal stationary peeling can be obtained. This nonstationary peeling apparatus is expected to be useful for evaluation of peel adhesion. © 1994 John Wiley & Sons, Inc.     

A coevolutionary approach to adapt the genotype-phenotype map in genetic algorithms

This article introduces a coevolutionary approach to genetic algorithms (GAs) for exploring not only within a part of the solution space defined by the genotype-pheno-type map, but also the map itself. In canonical GAs with a fixed map, how large an area of the solution space can be covered by possible genomes, and consequently how better solutions can be found by a GA, rely on how well the geotype-phenotype map in designed, but it is difficult for designers of the algorithms to design the map without a priori knowledge of the solution space. In the proposed algorithm, the genotype-phenotype map is improved adaptively during the search process for solution candidates. It is applied to 3-bit deceptive problems such as of typical combinatorial optimazation problems. These are well known because their difficulty for GAs can be controlled by the genotype-phenotype map, and this shows a fairly good performance compared with a conventional GA. This work was presented in part at the Sixth International Symposium on Artificial Life and Robotics, Tokyo, January 15–17, 2001.