Antibody Responses Raised against a Conformational V3 Loop Peptide of HIV-1

The amino acid sequence of the principal neutralizing determinant (PND) of 224 cases of human immunodeficiency virus type 1 (HIV-1) was determined and the most frequently occurring sequence was used as a peptide antigen for studying virus-specific antibody responses. In our present study, a linear peptide of the most frequent PND was first synthesized and then oxidized to create a disulfide-bridged loop conformation. Then, in order to construct a macromolecular structure for the purpose of increasing anti-genicity, the synthetic peptide was conjugated to a core peptide. We compared the immunogenicity of the disulfide-bridged loop PND peptide antigen (AG4) and the linear PND peptide antigen (AG5). After immunizing rabbits 5 and 6 times with both peptides, the results obtained using ELISA revealed that AG4 (conformational-loop type) was more capable of inducing a high titer of antigen-specific antibodies than was AG5 (linear type). Despite an amino acid sequence homology of 72%, a 1:8 dilution of serum raised against AG4 inhibited 81.9% of HIV-1_IIIB-mediated cell fusion, suggesting that conformational V3 loop peptide is able to elicit an antibody response which is strongly HIV-1-specific.     

Direct HPLC separation of enantiomers of pantoprazole and other benzimidazole sulfoxides using cellulose-based chiral stationary phases in reversed-phase mode

A direct, isocratic, and simple reversed-phase HPLC method was described for the separation of enantiomers of the proton pump inhibitor, rac-pantoprazole (PAN) using cellulose-based chiral stationary phases (Chiralcel OD-R and Chiralcel OJ-R). Some structurally related chiral benzimidazole sulfoxides, rac-omeprazole (OME) and raclansoprazole (LAN), were also studied. Chiralcel OJ-R was successful in the resolution of enantiomers of rac-PAN and rac-OME, while Chiralcel OD-R was most suitable for resolving the enantiomers of rac-LAN. Highest enantioselectivity to rac-PAN and rac-OME was achieved on Chiralcel OJ-R by using acetonitrile as an organic modifier, whereas methanol afforded better resolution of rac-LAN on Chiralcel OD-R than acetonitrile. Increases in buffer concentration and column temperature decreased retention and did not improve the resolution of the enantiomers on both columns. Using a mixture of 50 mM sodium perchlorate solution and acetonitrile as a mobile phase at a flow rate of 0.5 ml/min, maximum separation factors of 1.26 and 1.13 were obtained for the enantiomers of rac-PAN and rac-OME using a Chiralcel OJ-R column, while maximum separation factor of 1.16 was obtained for the enantiomers of rac-LAN using a Chiralcel OD-R column. © 1995 Wiley-Liss, Inc.     

The breakpoint on 7p in a patient with t(6;7) and craniosynostosis is spanned by a YAC clone containing the D7S503 locus

We previously reported a patient with an apparently balanced t(6;7) translocation and craniosynostosis. We now demonstrate, by fluorescence in situ hybridization, that the yeast artificial chromosome clone 933_e_l from the Centre d'Etude du Polymorphisme Humain library harbouring the D7S503 locus spans the breakpoint on distal 7p. Recent reports have defined a candidate region for a Saethre-Chotzen craniosynostosis locus between the loci D7S513 and D7S516, a region that includes the D7S503 locus. Since the translocation carrier shows only some of the symptoms characteristic for the Saethre-Chotzen syndrome, it remains unresolved whether the gene disrupted by the translocation event is the only one causing craniosynostosis in this chromosomal region.     

A possible mechanism for the hypoxia-hypoglycemia-induced release of excitatory amino acids from cultured hippocampal astrocytes

In order to elucidate the mechanism of release of excitatory amino acid (EAA) induced by hypoxiahypoglycemia (in vitro ischemia) from cultured hippocampal astrocytes, we compared the EAA release by in vitro ischemia with those by other treatments. The EAA release induced by in vitro ischemia treatment was rapid and reversible. The amount of released aspartate was comparable to that of glutamate, although the endogenous content of aspartate was one sixth that of glutamate. High-K (100 mM) treatment and the addition of 5 mM NaCN induced a rapid EAA release and the glutamate release was much greater than aspartate. Addition of 5 mM iodoacetate, a glycolysis inhibitor, induced a slow EAA release, and the amount of released aspartate was much higher than that of glutamate. On the other hand, the in vitro ischemia treatment and the addition of 5 mM NaCN induced only 20% reduction in ATP content for initial 5 min, whereas the addition of 5 mM iodiacetate induced a marked reduction. Our data suggest that ischemia-induced EAA release from astrocytes is a complex process in which local energy failure, inhibition of glycolysis, and depolarization of the cell membrane are involved.Abbreviations used EAA excitatory amino acids - PEI polyethyleneimine - DMEM Dulbecco's modified Eagle medium - HKR Hepesbuffered Krebs-Ringer solution     

Dinucleotide repeat polymorphism in the human ceruloplasmin gene

We have identified a GT dinucleotide repeat polymorphism in intron 14 of the human ceruloplasmin gene. Observed heterozygosity for the polymorphism is 0.84.     

A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP⁺ or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 × 10^?19 mol/assay and 2.5 × 10^?19 mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP⁺ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimtric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x ? 0.04, r = 0.997 (n = 51) and y = 1.00x ? 0.03, r = 0.999 (n = 10), respectively.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of theλbio specialized transducing phage inEscherichia coli. Because mostλbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^? phenotype), we selectedλbio transducing phage by their Spi^? phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofλbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio andλ DNAs, except for a hotspot which accounts for 57% of UV-inducedλbio transducing phages and 77% of spontaneously inducedλbio transducing phages. The hotspot sites onE. coli andλ DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio andλ hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

A novel actin filament (F-actin)–binding protein with a molecular mass of ~205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.     

Enhancement of Apoptosis in Developing Chick Neural Retina Cells by Basic Fibroblast Growth Factor

Abstract: To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time- and dose-dependent manner. The effect was inhibited by anti-bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-α, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.