Cloned primed-lymphocyte-test reagents in the dissection of HLA-D.

Human T lymphocytes obtained as blasts on day 4 from a primary mixed leukocyte culture (MLC) were cloned in the presence of T cell growth factor (TCGF) and feeder cells. Parameters important in producing higher-specific-activity TCGF were evaluated; irradiation of the responding cells as well as removal of adherent cells or inclusion of indomethacin in the culture was important. In addition, the presence of an irradiated lymphoblastoid cell line (LCL) cell in the TCGF-producing system enhanced activity in the supernate. The long-term maintenance of progeny from clones was achieved by utilizing the LCL autologous with either the responding or sensitizing cells from the initial MLC as feeder cells. Under those conditions, clones could be expanded for 7 or more wk with the maintenance of PLT reactivity. Had all the cells in each clone been maintained for the full 7 wk, more than 1 X 10(10) cells could have been developed in each clone. The cloned reagents provide a higher degree of antigen-specific reactivity than do normal PLT cells. It is to be anticipated that as the requirements for cloning are made more stringent, including the recloning of the cells, these reagents will aid greatly in the dissection of the complexity attendant to HLA-D.

Membrane adhesion in peripheral myelin: good and bad wraps with protein P0.

Recent molecular models and crystallographic analysis of the major protein of peripheral myelin have provided new insights into the molecular basis of membrane adhesion in myelin. These studies have proved useful in understanding the molecular basis of clinical phenotypes in certain demyelinating neuropathies.

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice.

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.

X-ray diffraction analysis of scrapie prion: intermediate and folded structures in a peptide containing two putative alpha-helices.

Small proteinaceous infectious particles called prions cause certain neurodegenerative diseases in human and animals. Limited proteolysis of infectious scrapie prions PrP(Sc) yields an N-truncated polypeptide termed PrP 27-30, which encompasses residues 90 to 231 of PrP(Sc) and which assembles into 100 to 200 A wide amyloid rods. It has been hypothesized that the infectious prion is converted from its non-infectious cellular form (PrP(C)) by means of an alpha-helical to beta-sheet conformational change. Secondary structure analysis, computer modeling, and structural biophysics methods support this hypothesis. Residues 90 to 145 of PrP, which contain two putative alpha-helical domains H1 and H2, may be of particular relevance to the disease pathogenesis, as C-terminal truncation at residue 145 was found in a patient with an inherited prion disease. Moreover, our recent X-ray diffraction analysis suggests that the peptide consisting of these residues (designated SHa 90-145) closely models the amyloidogenic beta-sheet core of PrP. In the current study, we have analyzed in detail the X-ray diffraction patterns of SHa 90-145. Two samples were examined: one that was dehydrated under ambient conditions whilst in an external magnetic field (to induce fibril orientation), and another that was sealed after partial drying. The dried, magnetically oriented sample showed a cross-beta diffraction pattern in which the fiber axis (rotation axis) was parallel to the H-bonding direction of the beta-sheets. The major wide-angle peaks indicate the presence of approximately 40 A wide beta-crystallites, which constitute the protofilament. Each crystallite is composed of several orthogonal unit cells, normal to the fiber (a-axis) direction, having lattice constants a = 9.69 A, b = 6.54 A, and c = 18.06 A. Electron density maps were calculated by iterative Fourier synthesis using beta-silk as an initial phase model. The distribution of density indicated that there were two types of beta-sheet, suggesting that larger and smaller side-chains localized to different sheets. This would arise from folding of the polypeptide in which there are turns in the middle of both the H1 and H2 domains. A monoclinic macrolattice, with a = 9.61 A, b = c = 52.99 A and alpha = 114.6 degrees, was found to index all the reflections, including those in the low-angle region. This suggests that the beta-crystallites are nearly hexagonally packed. To account for the approximately 100 A wide fibers visualized by negative staining in the electron microscope, the beta-crystallites would be arranged in 4-mers. The partially dried sample showed a sharp 4.7 A reflection (from H-bonding) and five broad peaks superimposed on monotonically decreasing diffuse scattering. This solution-like scattering was modeled by an anisometric rectangle with a thickness comparable to a singe beta-chain. The structure, which occurred during dehydration, could be a transient in the alpha-helical to beta-sheet conversion, suggesting that formation of hydrogen bonding precedes the inter-sheet interaction and assembly into the amyloid of scrapie prion.

Structural changes in a hydrophobic domain of the prion protein induced by hydration and by ala-->Val and pro-->Leu substitutions.

X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Sträussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.

X-ray diffraction of scrapie prion rods and PrP peptides.

Certain neurodegenerative diseases in humans and animals are caused by small proteinaceous infectious particles called prions. Limited proteolysis and detergent extraction of the prions containing PrPSc generate prion rods that are composed of a polypeptide having an apparent molecular mass of 27 to 30 kDa. This polypeptide, termed prion protein PrP 27-30, has a ragged N terminus that begins at about residue 90, but retains scrapie infectivity. Moreover, the findings in a patient having an inherited prion disease of a truncated PrP with its C terminus at residue 145 suggest that the residues 90 to 145 may be of particular importance in the pathogenesis of prion diseases. To determine the three-dimensional organization of prion rods and to identify the core region involved in amyloid formation, we recorded X-ray diffraction patterns from rods purified from scrapie-infected Syrian hamster (SHa) brains which contain PrP 27-30, and from synthetic SHaPrP peptides. Three peptides were studied corresponding to residues 113 to 120 (peptide A8A, an octamer composed of glycines and alanines), 109 to 122 (H1, the first predicted alpha-helical region of PrPC), and 90 to 145 (a 56 residue peptide containing both H1 and the second predicted alpha-helical region of PrPC, H2). Electron microscopy, carried out in parallel with the X-ray measurements, revealed that all the samples formed linear polymers which were approximately 60 to approximately 200 A wide, with fibrillar or ribbon-like morphology. Gels and dried preparations of prion rods gave X-ray patterns that indicated a beta-sheet conformation, in which the hydrogen bond distance was 4.72 A and the intersheet distance was 8.82 A. For the three PrP peptides, the intersheet spacings varied widely, owing to the side-chains of the residues involved in the formation of the beta-sheet interactions, i.e., 5.13 A for A8A, 5.91 A for lyophilized H1, 7.99 A from solubilized and dried H1 and 9.15 A for the peptide SHa 90-145. The intersheet distance of PrP 27-30 was thus within the observed range for the peptides, and suggests that the amyloidogenic core of PrP is closely modeled by the peptide SHa 90-145.

Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.

Vectors were constructed that allow foreign peptides to be expressed in Escherichia coli as fusion proteins. The peptides are fused to the C terminus of maltose-binding protein (MBP), which allows them to be purified by the MBP's affinity to cross-linked amylose (starch). The fusion protein can be directed to the periplasm by including the leader sequence from the phoA gene on the vector.

HLA-D clusters associated with DR4 in the Japanese population.

A study of HLA-D clusters associated with DR4 was performed in the Japanese population. These clusters consist of DYT, DKT2, DB3, and Dw4. Forty-two Japanese typed as DR4 were investigated, and it was found that 17 (40.5%) were DYT, 7 (16.7%) DKT2, 7 (16.7%) DB3, and 4 (9.5%) Dw4.

Increase of IgA-bearing peripheral blood lymphocytes in families of patients with IgA nephropathy.

IgA-bearing peripheral blood lymphocytes, serum IgA, urinary sediments and HLA types of patients with IgA nephropathy and members of their families were examined to elucidate whether some familial factors might be related to the development of IgA nephropathy. Ten patients with IgA nephropathy, 31 family members and 36 age-matched healthy persons were examined. All families included certain members with increased amounts of IgA-bearing peripheral blood lymphocytes. The pattern of the emergence of family members with increased IgA-bearing lymphocytes was vertical. Some family members who had increased IgA-bearing lymphocytes showed microhematuria at the time of the study. There was no significant correlation between the amounts of IgA-bearing peripheral blood lymphocytes and levels of serum IgA. HLA types of the ten patients did not show significant deviation from those in the general population. It is suggested that the measurement of IgA-bearing peripheral blood lymphocytes among family members is useful for the screening of patients with IgA nephropathy.

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.

Analysis of x-ray diffraction patterns from amyloid of biopsied vitreous humor and kidney of transthyretin (TTR) Met30 familial amyloidotic polyneuropathy (FAP) patients: axially arrayed TTR monomers constitute the protofilament.

Familial amyloidotic polyneuropathy (FAP) is characterized by deposits of amyloid fibers in which the major protein component is transthyretin (TTR). Nearly fifty mutations have been reported for the TTR in hereditary FAP. Protein crystallography of mutant TTRs has shown that the molecular structures of the variant molecules are similar to those found in the wild type. On this basis, the FAP fibers were initially proposed to consist of native-like TTR tetramers. In the current paper, we used x-ray fiber diffraction to study the structure of FAP fibers from biopsy samples of vitreous humor and kidney. The reflections of the vitreous sample showed a cross-beta diffraction pattern. All the meridional reflections were indexed by a one-dimensional, 29 A-period lattice, and the equatorial reflections were indexed by an apparent one-dimensional 67 A-period lattice. The x-ray intensity distribution indicated that the unit structure, which is similar to a TTR monomer, is composed of a pair of beta-sheets consisting of four hydrogen-bonded beta-chains per sheet, with the beta-chains oriented approximately normal to the fiber axis. The axial disposition of these units, with a 29 A-period, constitutes the protofilament; and a tetrameric lateral assembly of the protofilaments containing the core domain of the approximately 20 A-wide beta-sheet structure constitutes the FAP amyloid fiber. An inter-fiber separation of 75 A in these concentrated samples accounts for the apparent one-dimensional lattice perpendicular to the fiber axis. In the delipidated kidney FAP sample, the diffraction pattern indicated a pair of beta-sheets, suggesting that the protofilament structure in kidney is similar to that in vitreous humor. In the non-delipidated sample the successive sharp reflections indexed to a one-dimensional, 48.9 A-lattice, and the electron density projection showed a density elevation at the center of a lipid bilayer. This suggests that lipid may be associated with the monomeric TTR in the kidney FAP protofilament.

Histidine residues underlie Congo red binding to A beta analogs.

The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (A beta) in terms of binding affinity and number of sites was quantitated from absorption spectroscopy (at 200-700 nm) by measuring the concentration of CR bound (CR-B) to AD A beta assemblies as a function of CR concentration and pH in 80% ethanol. The rationale for the use of this high concentration of ethanol derives from its use in histological screens for amyloid in tissue sections. Moreover, free CR can be separated from bound CR by filtration in ethanolic but not aqueous medium. The A beta analogs studied here included: (1) peptides having different lengths: A beta1-40, A beta11-28, A beta13-28, A beta19-28, A beta11-25; (2) wildtype, control sequences of A beta1-40 and sequences having different natural amino acid substitutions: primate Pr1-40, rodent Ro1-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Du1-40, primate reverse sequence Pr40-1; and (3) A beta11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A beta1-40 fibrils in buffer were indistinguishable from those in buffered ethanolic medium. For all amyloid analogs except A beta19-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a function of increasing pH. The decrease was steepest at about pH 5 and became zero above pH 7. For analogs having the same number of histidines, CR-B fell on the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was parameterized by the stoichiometry of dye to the sites, the number of histidines per molecule, the binding dissociation constant Kd, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-titration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5.0-5.5, which was similar to the expected pK of histidine in low dielectric medium (80% ethanol), and the Kd's were 2.8-5.9 microM. That histidine residues underlie CR binding in A beta amyloid is consistent with previous findings that A beta peptides sediment as fibrillar assemblies at pH-3-7 and bind Congo red over the same pH range in aqueous medium. Further, the conformation near the binding motif His13-His14-Gln15-Lys16 in A beta assemblies is not greatly altered in 80% ethanol.

X-ray scattering from a discrete helix with cumulative angular and translational disorders.

X-ray scattering from a discrete helix possessing cumulative angular and translational disorders is studied by the Barakat model [Barakat (1987). Acta Cryst. A43, 45-49] assuming Gaussian distributions for random rotations and translations between subunits. This model is found to be identical to the paracrystalline model of the second kind. The intensity function shows that the intensity maximum decreases with increase in the Bessel order, whereas the peak breadth in the fiber direction and the intensity minimum increase.

Stabilization of sulfisomidine tablets by use of film coating containing UV absorber: Protection of coloration and photolytic degradation from exaggerated light.

The effect of model polymer coating films of vinyl acetate, containing oxybenzone as a UV absorber, on the coloration and photolytic degradation of simple sulfisomidine tablets was examined to attempt stabilization of photosensitive solid dosage forms. Coloration of the tablet surface was followed by the tristimulus colorimetric method in the fading tester equipped with a mercury vapor lamp. Photolytic degradation in the UV region was investigated by a new method for measuring the absorption spectra of a crystal sample in the gas phase, i.e., the semi-integral attenuance spectra. Two parameters of a film, thickness and concentration of the UV absorber, were varied at every exposure. These physical and chemical changes are discussed in relation to light transmission properties of films.

Implications of the sequence similarities between tau and myelin basic protein.

The minor myelin basic protein (MBP) isoforms with M(r) 21.5 and 17 kDa and the cytoskeletal proteins actin and tubulin are enriched in an interlamellar junctional specialization within central nervous system (CNS) myelin, the radial component (RC). To pursue the notion that there are specific interactions between these constituents, we searched for sequences in MBP that are homologous to sequences in the tubulin-binding protein tau. We found that the sequence motifs that are homologous to the phosphorylation and tubulin binding sites of tau (-RSP- and -KPGFG-) are also within the exon 2 and 6-encoded peptides of MBP. The -KPGFG- motif is unique to MBP when compared to other myelin proteins, and is highly conserved in the MBPs among vertebrate species. The physicochemical properties of the MBP and tau peptides that contain these sequences and their predicted secondary structures suggest that the peptides containing these motifs are hydrophilic and folded largely in turn and coil. This implies that the motifs are located at the protein surface where they would be accessible for interactions with other components of proteins or lipids. We propose that these putative phosphorylation and tubulin-binding sites in MBP may play functional roles in CNS myelin that are analogous to their roles in tau.

Risk assessment of drinking water in a reservoir contaminated by PAH's originated from road traffic.

The loads of Polycyclic Aromatic Hydrocarbons (PAHs) originating from road traffic were measured and in units of per vehicle per meter was estimated as follows: 0.07 ng/veh.m for Benzo[a]pyrene, and 0.83 ng/veh.m for Dibenzanthracene and so on, and 5.77 ng/veh.m for total PAHs. This unit is applied to risk estimation of drinking water in a reservoir where it is planned to construct a new high way the near future, and the concentration in the reservoir water is estimated to be 3.3-101 ng/l for individual PAH's. Assuming standard oral exposure to PAHs in raw water for drinking water supply, the estimated lifetime risk of carcinogenesis was less than 1 in 10(6), which is not considered significant.

Asymptomatic hemobilia with idiopathic thrombocytopenic purpura.

A severe but asymptomatic anemia developed in a patient with idiopathic thrombocytopenic purpura. Urgent upper gastrointestinal endoscopy disclosed massive bleeding from the papilla of Vater and we inferred that the anemia was caused by hemobilia. The bleeding point could not be detected, but the bleeding was stopped with conservative therapy. Although several cases of hemobilia have been reported in patients with bleeding tendency, this is the first case to be reported in a patient with idiopathic thrombocytopenic purpura.

Further study on HLA-A, B, C, D, DR and haplotype antigen frequencies in psoriasis vulgaris.

HLA-A, B, C, D, and DR antigens in 40 Japanese patients with psoriasis vulgaris were analysed using the antisera obtained from the Seventh International Histocompatibility Workshop. The most deviant HLA antigens in psoriasis were found to be HLA-Cw6 and DRw7. by observing the HLA-A, B, C and DR antigens en bloc in psoriasis, it is shown that the statistically significantly associated haplotype antigens were HLA-A1, B13, Cw6 and DRw7, and A1, B37, Cw6 and DRw6 respectively. No HLA-Dw11 (LD17) antigen was associated with the disease.

[Enzyme-linked immunosorbent assay using monoclonal antibodies for direct serotyping of enteric adenoviruses in feces].

We were prepared three monoclonal antibodies in which the monoclone 12D was type specific for Adenovirus 40 (Ad40), 1F was type specific for Ad41 and 15D was group specific for Ads. For identification of enteric adenoviruses (EAd) in stool specimens, enzyme-linked immunosorbent assay (ELISA) test using monoclonal antibodies was developed. Results of identification by the ELISA tests using monoclonal antibodies to EAd on 15 fecal samples in which Ad particles were found by electron microscopy showed complete coincidence to those of Sma 1 restriction endonuclease cleavage. From these results, the ELISA tests employing EAds type specific monoclonal antibodies proved to be specific and this was a rapid technique for laboratory diagnosis of EAd in fecal specimens of viral gastroenteritis. Fifty-eight fecal samples with Ad particles positive by EM were serotyped by the ELISA using monoclonal antibodies. Eleven fecal samples were identified as Ad40, 25 as Ad41, 1 as double infection with Ad40 and Ad41, and 4 as non-EAd. These results indicated that Ad41 was more dominant than Ad40 during April, 1986 to January, 1989 in Matsuyama city.