Identification of alpha-fodrin as a candidate autoantigen in primary Sjögren's syndrome.

It is unclear whether organ-specific autoantigens are critical for the development of primary Sjögren's syndrome (SS). A 120-kilodalton organ-specific autoantigen was purified from salivary gland tissues of an NFS/sld mouse model of human SS. The amino-terminal residues were identical to those of the human cytoskeletal protein alpha-fodrin. The purified antigen induced proliferative T cell responses and production of interleukin-2 and interferon-gamma in vitro. Neonatal immunization with the 120-kilodalton antigen prevented the disease in mice. Sera from patients with SS reacted positively with purified antigen and recombinant human alpha-fodrin protein, whereas those from patients with systemic lupus erythematosus and rheumatoid arthritis did not. Thus, the immune response to 120-kilodalton alpha-fodrin could be important in the initial development of primary SS.

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Structure of the B-DNA decamer C-C-A-A-C-G-T-T-G-G and comparison with isomorphous decamers C-C-A-A-G-A-T-T-G-G and C-C-A-G-G-C-C-T-G-G.

The crystal structure of the DNA decamer C-C-A-A-C-G-T-T-G-G has been solved to a resolution of 1.4 A, and is compared with the 1.3 A structure of C-C-A-A-G-A-T-T-G-G and the 1.6 A structure of C-C-A-G-G-C-C-T-G-G. All three decamers crystallize isomorphously in space group C2 with five base-pairs per asymmetric unit, and with decamer double helices stacked atop one another along the c axis in a manner that closely approximates a continuous B helix. This efficient stacking probably accounts for the high resolution of the crystal data. Comparison of the three decamers reveals the following. (1) Minor groove width is more variable than heretofore realized. Regions of A.T base-pairs tend to be narrower than average, although two successive A.T base-pairs alone may not be sufficient to produce narrowing. The minor groove is wider in regions where BII phosphate conformations are opposed diagonally across the groove. (2) Narrow regions of minor groove exhibit a zig-zag spine of hydration, as was first seen in C-G-C-G-A-A-T-T-C-G-C-G, whereas wide regions show two ribbons of water molecules down the walls, connecting base edge N or O with sugar O-4' atoms. Regions of intermediate groove width may accommodate neither pattern of hydration well, and may exhibit a less regular pattern of hydration. (3) Base-pair stacking is virtually identical at equivalent positions in the three decamers. The unconnected step from the top of one decamer helix to the bottom of the next helix is a normal helix step in all respects, except for the absence of connecting phosphate groups. (4) BII phosphate conformation require the unstacking of the two bases linked by the phosphate, but do not necessarily follow as an inevitable consequence of unstacking. They have an influence on minor groove width as noted in point (1) above. (5) Sugar ring pseudorotation P and main-chain torsion angle delta show an excellent correlation as given by the equation: delta = 40 degrees cos (P + 144 degrees) + 120 degrees. Although centered around C-2'-endo, the conformations in these B-DNA helices are distributed broadly from C-3'-exo to O-4'-endo, unlike the tighter clustering around C-3'-endo observed in A-DNA oligomer structures.

Analysis of local helix geometry in three B-DNA decamers and eight dodecamers.

Local variations in B-DNA helix structure are compared among three decamers and eight dodecamers, which contain examples of all ten base-pair step types. All pairwise combinations of helix parameters are compared by linear regression analysis, in a search for internal relationships as well as correlations with base sequence. The primary conclusions are: (1) Three-center hydrogen bonds between base-pairs occur frequently in the major groove at C-C, C-A, A-A and A-C steps, but are less convincing at C-C and C-T steps in the minor groove. The requirements for large base-pair propeller are (1) that the base-pair should be A.T rather than G.C, and (2) that it be involved in a major groove three-center hydrogen bond with the following base-pair. Either condition alone is insufficient. Hence, a large propeller is expected at the leading base-pair of A-A and A-C steps, but not at A-T, T-A, C-A or C-C steps. (2) A systematic and quantitative linkage exists between helix variables twist, rise, cup and roll, of such strength that the rise between base-pairs can hardly be described as an independent variable at all. Two typical patterns of behavior are observed at steps from one base-pair to the next: high twist profile (HTP), characterized by high twist, low rise, positive cup and negative roll, and low twist profile (LTP), marked by low twist, high rise; negative cup and positive roll. Examples of HTP are steps G-C, G-A and Y-C-A-R, where Y is pyrimidine and R is purine. Examples of LTP steps are C-G, G-G, A-G and C-A steps other than Y-C-A-R. (3) The minor groove is especially narrow across the two base-pairs of the following steps: A-T, T-A, A-A and G-A. (4) In general, base step geometry cannot be correlated solely with the bases that define the step in question; the two flanking steps also must be taken into account. Hence, local helix structure must be studied in the context, not of two base-pairs: A-B, but of four: x-A-B-y. Calladine's rules, although too simple in detail, were correct in defining the length of sequence over which a given perturbation is expressed. Whereas ten different two-base steps are possible, allowing for the identity of complementary sequences, there are 136 different four-base steps. Only 33 of these 136 four-base steps are represented in the decamer and dodecamer structures solved to date, and hence it is premature to try to set up detailed structural algorithms. (5) The sugar-phosphate backbone chains of B-DNA place strong limits on sequence-induced structural variation, damping down most variables within four or five base-pairs, and preventing purine-purine anti-anti mismatches from causing bulges in the double helix. Hence, although short-range sequence-induced deformations (or deformability) are observed, long-range deformations propagated down the helix are not to be expected.

Structure of a B-DNA decamer with a central T-A step: C-G-A-T-T-A-A-T-C-G.

The X-ray crystal structure analysis of the decamer C-G-A-T-T-A-A-T-C-G has been carried out to a resolution of 1.5 A. The crystals are space group P2(1)2(1)2(1), cell dimensions a = 38.60 A, b = 39.10 A, c = 33.07 A. The structure was solved by molecular replacement and refined with X-PLOR and NUCLSQ. The final R factor for a model with 404 DNA atoms, 108 water molecules and one magnesium hexahydrate cation is 15.7%. The double helix is essentially isostructural with C-G-A-T-C-G-A-T-C-G, with closely similar local helix parameters. The structure of the T-T-A-A center differs from that found in C-G-C-G-T-T-A-A-C-G-C-G in that the minor groove in our decamer is wide at the central T-A step rather than narrow, and the twist angle of the T-A step is small (31.1 degrees) rather than large. Whereas the tetrad model provides a convenient framework for discussing local DNA helix structure, it cannot be the entire story. The articulated helix model of DNA structure proposes that certain sequence regions of DNA show preferential twisting or bending properties, whereas other regions are less capable of deformation, in a manner that may be useful in sequence recognition by drugs and protein. Further crystal structure analyses should help to delineate the precise nature of sequence-dependent articulation in the DNA double helix.

The crystal structure of the trigonal decamer C-G-A-T-C-G-6meA-T-C-G: a B-DNA helix with 10.6 base-pairs per turn.

The B-DNA decanucleotide C-G-A-T-C-G-6meA-T-C-G has been crystallized under the same conditions used earlier for C-G-A-T-C-G-A-T-C-G, but is found to adopt a new trigonal P3(2)21 packing mode instead of the expected orthorhombic P2(1)2(1)2(1) form. Unit cell dimensions a = b = 33.38 A, c = 98.30 A, gamma = 120 degrees, imply ten base-pairs or one complete decamer double helix per asymmetric unit. The 2282 two-sigma data to 2.0 A refine to R = 17.2% with 45 water molecules, 1.5 hexavalent hydrated magnesium complexes, and 0.5 chloride ion per asymmetric unit. Neighboring helices interlock backbone chains and major grooves, crossing at an angle of 120 degrees in a manner that yields an excellent model for a Holliday junction. Local helix parameters differ markedly in the trigonal and orthorhombic structures, with the trigonal helix exhibiting behavior closer to that expected of B-DNA in solution. The trigonal form has an average of 10.6 base-pairs per turn, in contrast to 9.7 base-pairs per turn in the orthorhombic cell. A comparison of all known B-DNA decamer and dodecamer crystal structure analyses indicates that, the greater the cell volume per base-pair (and hence the more open the crystal structure), the closer the mean helix twist approaches an expected 10.6 base-pairs per turn.

Simple suturing of the nasal septum using the Maniceps septum stitch device.

OBJECTIVE: To present the utility of the recently introduced Maniceps septum stitch device for suturing of the nasal septum. METHODS: This paper describes nasal septum suturing techniques using the Maniceps septum stitch device following septoplasty to prevent post-operative complications such as haematoma and nasal septum perforation. CONCLUSION: Nasal septum suturing using the Maniceps septum stitch device appears to be a safe and easy surgical procedure to help prevent post-operative complications and may reduce the incidence of nasal septum perforation following septoplasty.

Electrically induced ambipolar spin vanishments in carbon nanotubes.

Carbon nanotubes (CNTs) exhibit various excellent properties, such as ballistic transport. However, their electrically induced charge carriers and the relation between their spin states and the ballistic transport have not yet been microscopically investigated because of experimental difficulties. Here we show an electron spin resonance (ESR) study of semiconducting single-walled CNT thin films to investigate their spin states and electrically induced charge carriers using transistor structures under device operation. The field-induced ESR technique is suitable for microscopic investigation because it can directly observe spins in the CNTs. We observed a clear correlation between the ESR decrease and the current increase under high charge density conditions, which directly demonstrated electrically induced ambipolar spin vanishments in the CNTs. The result provides a first clear evidence of antimagnetic interactions between spins of electrically induced charge carriers and vacancies in the CNTs. The ambipolar spin vanishments would contribute the improvement of transport properties of CNTs because of greatly reduced carrier scatterings.

Neonatal lupus erythematosus: maternal IgG antibodies bind to a recombinant NH2-terminal fusion protein encoded by human alpha-fodrin cDNA.

IgG antibodies to a cleavage product of alpha-fodrin (120 kDa alpha-fodrin) have recently been identified as organ-specific autoantibodies in primary Sjögren's syndrome. In this study, we examined seroreactivity of mothers and infants with neonatal lupus erythematosus (NLE) to a recombinant NH2-terminal protein (120 kDa alpha-fodrin) of human alpha-fodrin. Serum samples were collected during the perinatal period in seven pregnancies of five mothers delivering offspring with NLE. Anti-120 kDa alpha-fodrin antibodies were identified by immunoblotting in six of seven perinatal maternal sera of offspring with NLE: one of two congenital heart block offspring and all five offspring with cutaneous NLE. These antibodies were placentally transmitted to infants. One of the five mothers had primary Sjögren's syndrome, and four were asymptomatic. One asymptomatic mother did not demonstrate anti-120 kDa alpha-fodrin activity at the time of the first delivery of a congenital heart block infant, but was found to be positive at the time of subsequent delivery of a second child with cutaneous NLE. We propose that maternal antibodies to 120 kDa alpha-fodrin may be an additional serologic marker for the risk of NLE in anti-Ro/SS-A positive women.

Synthesis and secretion of biologically active mouse interferon-beta using a Bacillus subtilis alpha-amylase secretion vector.

Mouse interferon-beta (IFN-beta) cDNA, whose signal sequence had been removed by BAL 31 digestion, was introduced into a Bacillus subtilis secretion vector constructed by using the promoter and signal sequence of the B. subtilis alpha-amylase gene. The resultant chimeric plasmids were transferred into B. subtilis 207-25. Four kanamycin-resistant transformants were selected by both colony hybridization and a new immunoblot method for secretory proteins. They secrete the proteins which cross-react with sheep anti-mouse IFN-beta serum into the culture medium. One of them expressed a high IFN-beta activity as assayed by the L cell and vesicular stomatitis virus system, while the other three showed weak or little IFN activities. Based on our previous study [Ohmura et al., Nucl. Acids Res. 12 (1984) 5307-5319], it was suggested that the secreted IFN molecules are hybrid proteins in which the NH2-terminal region consists of part of the alpha-amylase signal peptide. Nucleotide sequence analysis revealed that plasmid pTUB502, which expressed high IFN activity, is joined to the mouse IFN-beta gene from the codon position 6 of its mature protein. The other three plasmids, pTUB506, pTUB509, and pTUB519, contain the mouse IFN-beta gene from the codon positions 3, 1, and -5, respectively. The NH2-terminal region of the mouse IFN-beta seems to be closely related to its biological activity.

Increased serum remnant lipoproteins in patients with apolipoprotein E7 (apo E Suita).

Apolipoprotein (apo) E7 was originally identified by Yamamura et al. in subjects with atherosclerotic cardiovascular diseases (J. Clin. Invest. 1984;74:1229). However, the lipoprotein abnormalities associated with apo E7 phenotype have not been elucidated. In the current study, to clarify the physiological roles of apo E7, lipoprotein abnormalities were studied in 12 apo E7 heterozygotes. A total of seven subjects were hyperlipidemic and five subjects were normolipidemic. The apo E phenotype was apo E7/3 in 11 subjects and apo E7/4 in one subject. Polymerase chain reaction revealed that all of the subjects with apo E7 phenotype had the same mutation as that of apo E(Suita) as reported previously (J. Biochem. 1989;105:249). All the hyperlipidemic subjects were over 40 years of age and two of them also had and severe coronary heart disease. Ultracentrifugal analysis revealed that the cholesterol level both in very low density lipoprotein and in intermediate density lipoprotein (IDL) was substantially higher in hyperlipidemic apo E7 heterozygotes, compared with control subjects and that the IDL cholesterol was also increased even in normolipidemic apo E7 heterozygotes. Polyacrylamide gel electrophoresis of lipoproteins showed a midband, which implies the increase of remnant lipoproteins, in 11 subjects out of 12, irrespective of the presence or absence of hyperlipoproteinemia. In two cases, a broad beta pattern was observed similar to that seen in type III hyperlipoproteinemia. Dietary therapy was dramatically effective for the treatment of hyperlipidemia in patients with apo E7. These findings confirm that apo E is crucial for remnant lipoprotein metabolism and that apo E7 is related to the increase in serum remnant lipoproteins, which leads to hyperlipoproteinemia in association with obesity, aging and impaired glucose metabolism.

Characteristics of coronary artery disease and lipoprotein abnormalities in patients with heterozygous familial hypercholesterolemia associated with diabetes mellitus or impaired glucose tolerance.

Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of serum low density lipoprotein (LDL) cholesterol and premature coronary atherosclerosis. In order to elucidate the influence of abnormal glucose metabolism on the development of coronary artery disease (CAD) in FH patients, we examined the prevalence of CAD and characteristics of lipoprotein abnormalities in patients with heterozygous FH who were accompanied by diabetes mellitus (DM) or impaired glucose tolerance (IGT). The subjects of the present study were 150 patients with heterozygous FH, all over 40 years of age. Oral glucose tolerance tests demonstrated that 15 patients had DM and 27 had IGT. The combination of DM or IGT with FH was associated with a further increase in the prevalence of CAD (DM:IGT:normal glucose tolerance (N), 87:59:43%). Furthermore, the prevalence of the stenoses in the distal coronary arteries was significantly higher in the DM group than in the N group, while there was no significant difference in the prevalence of proximal and middle lesions. Serum triglyceride levels were significantly higher in the DM and IGT groups than in the N group (P < 0.01, DM versus N group; P < 0.01, IGT versus N group), while total cholesterol levels were not significantly different. When lipoproteins were analyzed by polyacrylamide gel electrophoresis, the frequency of midband appearance, which implies an increase in remnant lipoproteins, was significantly higher in the DM and IGT groups than in the N group (DM:IGT:N, 87:72:29%, P < 0.01, DM versus N group; P < 0.01, IGT versus N group). Ultracentrifugation analysis of lipoproteins revealed that intermediate density lipoprotein cholesterol was increased in DM and IGT groups compared with the N group. These data suggest that abnormal glucose metabolism may accelerate the development of CAD in FH patients due to an increase in atherogenic remnant lipoproteins in addition to high concentration of LDL. Special attention should be paid in the treatment of FH patients with impaired glucose metabolism, to avoid the advancement of coronary atherosclerosis.

Antiplatelet and antithrombotic effects of YM337, the Fab fragment of a humanized anti-GPIIb/IIIa monoclonal antibody in monkeys.

The antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 micrograms/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 micrograms/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation. Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 micrograms/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 +/- 5.2 min) or sodium ozagrel (9.4 +/- 3.0 min) compared with saline (13.3 +/- 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model. These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.

Studies on ribosomal ribonucleic acid from yeast. III. Secondary structure of 18 and 26S yeast ribosomal RNA's and their complex: circular dichroism and infrared analyses.

Circular dichroism (CD) and infrared (IR) spectroscopic analyses were performed of 26 and 18S yeast ribosomal RNA's (rRNA) and their specific complex, 30S RNA. The molecular ellipticity coefficients [thota] of 18, 26, and 30S rRNA's were 2.72, 2.63, and 2.65X10(4) degree-cm2/decimole at 264 nm, respectively. The base-pairing contents of 18, 26, and 30S rna's determined by iC), and 70% (32% AU, 38% GC), respectively. These results suggest that 18 and 26S rRNA have very similar secondary structures, and that 30S rna may have a slightly higher base-pairing content than the estimated sum of those of 18 and 26S rRNA's. The biological significance of this phenomenon is discussed in this report.

Human papillomavirus DNA in condylomata acuminata from Japanese males.

We examined condylomata acuminata from Japanese males for the presence of human papillomavirus (HPV) genomes by Southern blot hybridization. HPV 6/11-related DNA was found in 91% (32/35) of the condylomata. HPV 6a DNA was found in 40% (14/35), HPV 6c DNA in 6% (2/35), and HPV 11a DNA in 37% (13/35). HPV 6-related DNA, which had an unusual PstI-cleavage pattern, was detected in one sample. Types and subtypes of HPV DNA in the samples studied (HPV 6a, 6c, and HPV 11a DNA) were not correlated with the patients' ages nor outcomes of the disease. HPV 16 DNA was not detected in any condyloma acuminatum.

Functional expression of GABA rho 3 receptors in Xenopus oocytes.

Homomeric rat GABA rho 3 receptors were expressed in Xenopus oocytes, and their pharmacological profile was investigated electrophysiologically. GABA activated the rho 3 receptors with an EC50 value of 7.5 microM and a Hill coefficient of 1.6. The GABA-induced current was not antagonized by bicuculline (100 microM), but was blocked by picrotoxin (IC50: 0.68 microM for 100 microM GABA). The current was almost insensitive to pentobarbital, diazepam and a neurosteroid, 3 alpha-OH-DHP. Many of the pharmacological properties of the rho 3 subunit were similar to those of the previously reported rat rho 1 and rho 2 subunits and GABAC receptors.

Synthesis of fusion proteins of Epstein-Barr virus nuclear antigens in E. coli and their antigenicity.

Expression and yield in E. coli of a panel of fusion proteins containing various domains of Epstein-Barr virus nuclear antigens, EBNA-1, EBNA-2, EBNA-3, EBNA-4 and EBNA-6, were scrutinized. The antigenicity of the EBNA fusion proteins against human sera was examined. Monospecific antisera to the different EBNA domains were produced by immunizing guinea pigs and rabbits. An EBNA-6 fusion polypeptide was useful for separating anti-EBNA-6 antibody from human sera by immunoaffinity purification. The applications of the fusion proteins to clinical diagnosis are discussed.

Synergistic effect of aurintricarboxylic acid and triflavin in a photochemically induced thrombosis model in rats.

We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.

Effect of 3-hydroxymethylene-2-thioxopyrrolidine on growth of two species of mutans streptococci and their in vitro plaque formation.

3-Hydroxymethylene-2-thioxopyrrolidine (HMTP), the major product derived from radish mustard oil, was studied for its activity to inhibit the growth of mutans streptococci, their in vitro plaque formation and their glucan production. The minimum inhibitory concentration (MIC) (800-1600 mg/l) of HMTP at pH 7.0 was reduced to 200 mg/l by lowering the medium pH to 5.0. A dose-dependent inhibition of in vitro plaque formation was observed at 200-800 mg/l dose of HMTP. Production of water-insoluble glucan (WIG) was effectively inhibited by 45-98%, depending on HMTP dose (200-800 mg/l), while only 22% inhibition of water-soluble glucan (WSG) production was observed at an 800 mg/l dose.