Molecular characterization of novel reciprocal translocation t(6;14) in an Epstein-Barr virus-transformed B cell precursor.

An in vitro culture of FLEB14 cells, an Epstein-Barr virus-transformed B cell precursor containing the germ line immunoglobulin genes, gave rise to a uniclonally expanded variant, FLEB14 delta 3, which was rearranged at the immunoglobulin heavy-chain gene locus. Cytogenetic analysis showed that FLEB14 delta 3 had a novel reciprocal translocation, t(6;14)(q15;q32). Molecular cloning of the rearranged DNA fragments and determination of their nucleotide sequence revealed that the recombination event was reciprocal, imprecise, and nonhomologous and took place in the S mu region, like those found in Burkitt's lymphoma cells. We propose a molecular model to explain this genetic event which may be relevant to class switch recombination. The translocated sequence of chromosome 6 did not contain any known oncogenes, although the sequence is conserved among mammals. FLEB14 delta 3 did not show tumorigenicity.

Coordination of microtubules and the actin cytoskeleton is important in osteoclast function, but calcitonin disrupts sealing zones without affecting microtubule networks.

Bone-resorbing osteoclasts form sealing zones and ruffled borders toward the bone surface. The sealing zone consists of a ring-like alignment of F-actin dots and surrounds the ruffled border, from which protons are secreted into the bone surface. Vacuolar-type proton ATPase (V-ATPase) in osteoclasts is a ruffled border-associated enzyme responsible for the proton secretion. We studied the interaction between microtubules and the actin cytoskeleton in osteoclasts. Confocal microscopic observation revealed that osteoclasts on glass coverslips, dentine slices and Osteologictrade mark discs formed the ring-like structure of F-actin dots, and microtubules overlapped the top of the F-actin dots. Osteoclasts cultured on dentine formed resorption pits within 48 h. The treatment of osteoclasts with cytochalasin D, an F-actin-depolymerizing reagent, induced perturbation of the microtubules in osteoclasts on glass and inhibited their pit-forming activity on dentine in a dose-dependent and reversible manner. Conversely, nocodazole, a microtubule-depolymerizing reagent, disrupted sealing zones and inhibited pit-forming activity of osteoclasts in a dose-dependent and reversible manner. V-ATPase showed a tendency to be localized inside sealing zones in osteoclasts. Treatment of osteoclasts with calcitonin induced both disruption of sealing zones and dispersion of V-ATPase to the whole area of the cytoplasm within 60 min. The microtubule networks in osteoclasts remained unchanged for 60 min even in the presence of calcitonin. These results suggest that coordination of the actin cytoskeleton and microtubules is important in the function of osteoclasts, but calcitonin selectively affects the actin cytoskeleton and induces the dispersion of V-ATPase without causing significant changes in the microtubules.

Heterogeneities in ferritin dimers as characterized by gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques.

To understand the mechanism underlying the preferential dimerization of ferritin shells, we studied monomers and dimers from both horse spleen and recombinant horse L-apoferritin by using gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques. Our study of the kinetics of dimer-monomer dissociation that is produced by heating revealed the presence of at least two types of dimers, namely, weakly and strongly linked dimers with activation energies of 124 +/- 14 and 157 +/- 16 kJ/mol, respectively. Our study using thiol reagents indicated that the dimerization in horse spleen ferritin is partially mediated by disulfide bridges being formed between H-chains. Our analysis of the components that resulted from the dimer-monomer dissociation further clarified that these dimers form interdigitation structures. In summary, five types of dimers were identified in horse spleen apoferritin: reversible dimers with very weak interaction, non-sulfide dimers with weak interaction, non-sulfide dimers with strong interaction, disulfide dimers linked only by disulfide bridges, and disulfide dimers linked by disulfide bridges and having other interactions.

Circular dichroism and biochemical properties of the hepatitis B virus core antigen.

The hepatitis B virus (HBV) core antigen was purified by mild procedures, including hydroxyapatite column chromatography, with care taken to avoid the degradation of the particles. Circular dichroism (CD) of the HBV core particles in saline showed low intensities of negative ellipticities in the region dominated by amide bond absorption. Acid treatment of the particles induced a remarkable change in the CD spectrum, with the appearance of a positive extremum at about 208 nm. The amino acid composition and the COOH-terminal residue of the isolated core polypeptide (Mr 21,000-21,500) were shown to be essentially the same as those of the polypeptide deduced from the nucleotide sequences which had been proposed for the HBV core antigen by other laboratories. We failed to detect any NH2-terminal dansyl-derivatives from the core polypeptide by the dansyl-Edman method. We also showed by the method of fluorescein polarization that the core polypeptide conjugated with fluorescein isothiocyanate has an affinity for serum albumin. This may indicate a state of disassembled or non-assembled core polypeptide in sera.

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Electrophysiologic effects of sodium channel blockade on anisotropic conduction and conduction block in canine myocardium: preferential slowing of longitudinal conduction by flecainide versus disopyramide or lidocaine.

OBJECTIVES: The purpose of this study was to determine the effects of sodium channel blockade on anisotropic excitation propagation in the intact canine left ventricle. BACKGROUND: Anisotropic ventricular conduction- electric conductivity dependent on the myocardial fiber direction-is one of the important mechanisms of ventricular arrhythmia. However, the effects of sodium channel blockade, especially the differential effect of a subclass of this agent, on the anisotropic properties remain unknown. METHODS: In 28 anesthetized, open chest dogs, a small cannula was inserted into the left anterior descending coronary artery. Saline (control), disopyramide, lidocaine or flecainide was infused selectively into the cannula. An array of 64 epicardial electrodes was placed on the anterior surface of the ventricle. Activation time (AT) was measured along the longitudinal (L) and transverse (T) directions. RESULTS: High dose flecainide (100 microg/kg body weight per min) delayed the AT along the L direction markedly (mean [+/-SE] 227 +/- 38%, p < 0.02) and mildly (121 +/- 10%, p < 0.02) along the T direction in regular beats (p < 0.007, L vs. T). Lidocaine and disopyramide did not show direction-dependent prolongation of the AT on regular beats. When examined on premature beats, AT was delayed, depending on the coupling interval and the fiber direction when saline, flecainide or lidocaine was infused. The conduction blocks along the L direction were observed in three of seven dogs on regular beats after flecainide and ventricular fibrillation ensued in two of these three dogs. CONCLUSIONS: A peculiar slowing of L conduction by flecainide may relate to the character of proarrhythmia.

Glibenclamide attenuates peaked T wave in early phase of myocardial ischemia.

OBJECTIVES: ECG peaked T wave appears during the early phase of myocardial ischemia, but the underlying mechanisms remain unknown. The purpose of this study was to elucidate the role of ATP-sensitive K+ channel (KATP) in this ECG change. METHODS: In 12 anesthetized, open-chest dogs, the sinus node was crushed and the right atrium was paced at a cycle length of 400 ms. The left anterior descending coronary artery was abruptly occluded for 60 s before (control) and 15 min after an intravenous infusion of vehicle (n = 6) or glibenclamide (1 mg/kg, n = 6), a blocker of KATP. Forty-eight epicardial electrograms were simultaneously recorded from the anterior surface of the left ventricle. The potentials at 40, 80 and 120 ms from the J point were measured, and these points corresponded to the early, middle and late phases of the T wave, respectively. RESULTS: During the control occlusion, T wave increased time-dependently and the maximal T-wave change was noted at the end of 60 s of coronary occlusion. The extents of T-wave elevation at the early, mid and late T phases were 5.5 +/- 0.5, 7.3 +/- 0.8 and 11.7 +/- 1.8 mV, respectively, and these T-wave elevations were significantly reduced by 33 +/- 21%, 59 +/- 12% and 63 +/- 13%, respectively, after the pretreatment with glibenclamide but not with its vehicle. The % reductions of mid and late T by glibenclamide were significantly larger than that of early T wave (P < 0.05). CONCLUSIONS: An abrupt coronary occlusion accompanied peaked T wave as an early ECG wave change. As the extent of this T-wave elevation was attenuated by glibenclamide, the ischemia-induced alteration of ventricular repolarization can partly (60%) be explained by the modification of KATP activation.

Comparison of iterated single-step and Bayesian regressions on genomic evaluations for age at 100 kg in swine.

The effects of modified single-step genomic best linear unbiased prediction (ssGBLUP) iterations on GEBV and SNP were investigated using 85,388 age at 100 kg phenotypes from the BRF SA breeding program Landrace pure line animals, off-tested between 2002 and 2013. Pedigree data comprised animals born between 1999 and 2013. A total of 1,068 animals were assigned to the training population, in which all of them had genotypes, original and corrected age at 100 kg phenotypes, and weighted deregressed proof records. A total of 100 genotyped animals, with high accuracy age at 100 kg estimated breeding values, were assigned to the validation population. After applying the quality control workflow, a set of 41,042 SNP was used for the analysis. Standard and modified ssGBLUP, BayesCπ, and Bayesian Lasso were compared, and their predictive abilities were accessed by approximate true and GEBV correlations. Modified ssGBLUP iteration effects on SNP estimates and GEBV were relevant, in which assigned differential weights and shrinkage caused important losses on ssGBLUP predictive ability for age at 100 kg GEBV. Even though ssGBLUP accuracy can be equal or better than the compared Bayesian methods, additional gains can be obtained by correctly identifying the number of iterations required for best ssGBLUP performance.

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen.

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transformation and bovine i-active glycolipid (NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

Site-specific reactivities of cysteine residues in horse L-apoferritin.

Recombinant horse L-apoferritin and its mutants were used to compare the reactivities of two different cysteinyl residues with 7-fluoro-4-sulfamoyl-2,1,3-benzoxadiazole (ABD-F), p-chloromercuribenzoic acid (PCMB), N-(9-acridinyl)maleimide (NAM), and 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (NEM-TEMPO). ABD-F selectively reacted with cysteine 52 (C52), which is located on the inner surface of the peptide shell of apoferritin. In contrast, PCMB reacted only with cysteine 130 (C130), which is located at the 3-fold channels of the shell. NAM and NEM-TEMPO reacted with both C52 and C130.

Characterization of a rat tRNA gene cluster: comparison of nucleotide sequences of the gene for tRNALeu newly found in repeating units.

In the rat, DNA carrying a cluster of the genes for tRNAAsp, tRNAGly, and tRNAGlu, aligned in that order, is repeated about 10 times. Seven DNA clones corresponding to the independent repeating units were isolated from a rat gene library. Nucleotide sequence analysis of these clones revealed the presence of a fourth tRNA gene, the gene for tRNALeu, in the cluster. The tRNALeu gene is located about 600 base pairs (bp) upstream from the tRNAAsp gene and its polarity differs from those of the other three tRNA genes. Among the repeating units, the nucleotide sequence of tRNALeu is conserved to a relatively high degree.

Refractive-index-adjustable fillers for visible-light-cured dental resin composites: preparation of TiO2-SiO2 glass powder by the sol-gel process.

New fillers have been prepared for visible-light-cured (VL) dental resin composites with the refractive index adjustable to that of the resin phase. These SiO2 glass powders containing TiO2 up to 20 wt% were formed by heating to 1000 degrees C ground gels made from a mixture of Ti[OCH(CH3)2]4 and Si(OC2H5)4. With increasing TiO2 content, the refractive index of the prepared power increased linearly, while the optical transmittance at 467 nm decreased linearly. The experimentally formulated VL-cured resin composites, consisting of (TEGDMA and Bis-GMA) monomer mixture and TiO2-SiO2 glass filler, had greater transmittance when the refractive index of the filler matched that of the monomer mixture, resulting in a greater degree of monomer conversion upon irradiation with VL.

Analysis of photo-initiators in visible-light-cured dental composite resins.

Seven commercial visible-light-cured (VL) dental composite resins were analytically studied for identification of the photo-initiator consisting of photo-sensitizer and reducing agent. Gas-liquid chromatography (GC) was used for the determination of the dilute components extracted from the composite resin. Mass spectroscopy (GC-MS) was used for confirmation of the qualitative data obtained by GC. The results showed that all composite resins examined included camphorquinone (CQ) as a photo-sensitizer. The concentration of CQ in the resin phase, however, ranged from 0.17 to 1.03% w/w. The composite resin with hybrid-sized filler tended to have a higher concentration of CQ than did the micro-filled composite resin. As for the reducing agent, two out of seven brands contained dimethylaminoethyl methacrylate (DMAEMA), and one included dimethyl-p-toluidine (DMPTI). The mixing ratio between CQ and the amine in these three composite resins also varied. Another four brands did not contain either DMAEMA or DMPTI, and would utilize different reducing agents.

Phenanthrenes from Dendrobium plicatile.

From Dendrobium plicatile stems, three phenanthrenes were isolated. The structures are 2,5-dihydroxy-4,9,10-trimethoxyphenanthrene, 2,5-dihydroxy-4-methoxyphenanthrene and 2,5,9-trihydroxy-4-methoxy-9,10-dihydrophenanthrene.

Performances of antibiotic screening tests in determining the persistence of penicillin residues in ewe's milk.

Milk collected at 12-h intervals throughout 6 days from three groups of Manchega ewes (n = 12 per group), treated intramuscularly with beta-lactams (benzyl penicillin procaine, ampicillin, and amoxicillin, respectively), was evaluated for antibiotic residue persistence with four microbial inhibitor tests (BRT MRL, CH ATK P&S, Delvotest SP, and Eclipse 100ov) and one enzymatic test (Penzym 100). Antibiotic depletion time was established using a logistic regression model. A clear effect (P < 0.0001) of milking order on the response of all tests was observed with the three antibiotics, but no significant effects were found for milk yield. Except with Eclipse 100ov, positive tests were observed after the recommended withdrawal period of benzyl penicillin procaine (five milkings) from 2% (Delvotest SP) to 11% (CH ATK P&S). There were almost no positive responses beyond the withdrawal period (six milkings) of ampicillin, except for the Penzym 100 test (7%). Residues of amoxicillin were found to persist beyond the six milkings established as the withdrawal period, from 2.8% (Eclipse 100ov) to 72.4% (CH ATK P&S) of positive cases. Higher frequencies of doubtful cases were found with BRT MRL and Delvotest SP assays with the three beta-lactams. Positive and doubtful results could be obtained when milk samples from individual ewes were analyzed using BRT MRL, CH ATK P&S, Delvotest SP, and Penzym 100 tests, even if farmers follow the antibiotic withdrawal periods.

Studies on radiopaque composites containing ZrO2-SiO2 fillers prepared by the sol-gel process.

Radiopacity can facilitate diagnostic observations adjacent to dental composites. To produce radiopaque SiO2-based fillers, ZrO2-SiO2 powders containing up to 45wt% ZrO2 were manufactured by the sol-gel process. The radiopacity of experimental composites containing Bis-GMA+TEGDMA+CQ+DMAEMA monomer mixture (25 wt%) and ZrO2-SiO2 fillers (75 wt%) was evaluated and compared to that of human enamel and two commercial composites with different radiopacity. It became evident that the radiopacity of the experimental composites increased linearly with zirconium content and could match that of the control materials, while the composite containing 15wt% ZrO2-85wt% SiO2 filler possessed radiopacity analogous to that of human enamel. It was demonstrated here that the radiopacity of the composites could be precisely controlled by adjusting the ZrO2 content in ZrO2-SiO2 fillers. These radiopacity-adjustable composites have the potential to be very useful filling materials by enabling dentists to accurately distinguish secondary caries adjacent to the composite restorations.

Cutting effectiveness and wear of carbide burs on eight machinable ceramics and bovine dentin.

As a first approach in evaluating the feasibility of industrial machinable ceramics in dentistry, we performed weight-load-cutting tests on eight machinable ceramics and bovine dentin, using #1557 carbide burs driven by an air-turbine handpiece. While the transverse load applied to the bur was cyclically varied between 20 and 80 g, we measured the cutting speed (i.e., the steady-state handpiece speed during cutting) and the cutting volume. The greater the applied load, the more the cutting speed decreased and the cutting volume increased. The degree of this trend, however, differed among the workpieces. When dentin and mica-based glass ceramics were being cut, the cutting speed was moderately reduced, the cutting effectiveness of the bur remained high, and the wear of the bur was small. When other ceramics--such as AIN-based, Si3N4-based, and CaO.SiO2-based ceramics--were being cut, however, the cutting speed was less diminished, and the cutting efficiency of the bur was smaller and decreased rapidly, along with extensive wear of the bur. We speculate that mica-based glass ceramics could be used as the substitute for dentin in the pre-clinical cutting exercise, and that another potential use of machinable ceramics examined might be in the production of future machined dental prostheses.

Dipyridamole electrocardiography test for the detection of severe coronary artery stenoses.

To investigate the relationship between dipyridamole-induced ST depression and the severity of coronary artery stenosis, the dipyridamole injection test (D) at 0.568 mg/kg/4 min, and the symptom limited treadmill exercise test (T) were performed separately in 16 normal volunteers and 167 patients who underwent coronary arteriography [91 patients without myocardial infarction (non-MI group) and 76 patients with previous myocardial infarction (MI group)]. Standard 12-lead electrocardiogram and body surface mapping of 87 leads were recorded in both tests. None of the normal volunteers had significant ST depression (greater than or equal to 0.10 mV) in D or T. Regarding the non-MI group, D had as high an incidence of ST depression as T (83% vs 93%) in patients with maximal coronary stenosis greater than or equal to 90%, while in those with maximum coronary stenosis less than 90%, D had a lower incidence of ST depression than T (16% vs 48%, p less than 0.01). For the MI group, the incidence of ST depression was compared to the maximal coronary artery stenosis supplying the non-infarcted area. In patients with maximal coronary artery stenosis greater than or equal to 90%, D had as high an incidence of ST depression as T (71% vs 64%). While, D had a lower incidence of ST depression than T (19% vs 35%, p less than 0.05) in those with maximum coronary artery stenosis less than 90%. For the diagnosis of coronary artery stenosis of greater than or equal to 90%, D had as high sensitivity (non-MI group, 82% vs 93%; MI group, 71% vs 64%) and higher specificity (non-MI group, 84% vs 52%, p less than 0.01; MI group, 81% vs 65%, p less than 0.05) compared with T. This study demonstrated that dipyridamole ECG is a sensitive and specific test to detect severe coronary artery stenosis.

Abnormal distribution of anionic sites in the glomerular basement membrane in glomerulonephritis of dogs infected with Dirofilaria immitis.

Ultrastructural alteration of anionic sites (ASs) in the glomerular basement membrane (GBM) was studied in glomerulonephritis characterized by linear capillary IgG deposition in four dogs infected with Dirofilaria immitis and two normal control dogs using polyethyleneimine. ASs were identified as small dense particles distributed regularly in the lamina rara externa (LRE), but there were no ASs in the lamina densa (LD) of the GBM of the control dogs. In the glomeruli of the infected dogs, ASs were distributed regularly or irregularly in the thickened LD. ASs were in addition localized over the characteristic continuous bands of subendothelial dense deposits. The number of ASs of the LRE increased in all four infected cases as compared to the controls (p<0.01).