Prevalence and growth kinetics of Shiga toxin-producing Escherichia coli (STEC) in bovine offal products in Japan.

Recent epidemiological data suggest a link between the consumption of bovine offal products and Shiga toxin-producing Escherichia coli (STEC) infection in Japan. This study thus examined the prevalence of STEC in various types of these foods. PCR screened 229 bovine offal products for the presence of Shiga toxin (stx) gene. Thirty-eight (16·6%) samples were stx positive, of which eight were positive for rfbE(O157) and three were positive for wzy(O26). Four O157 and one O26 STEC isolates were finally obtained from small-intestine and omasum products. Notably, homogenates of bovine intestinal products significantly reduced the extent of growth of O157 in the enrichment process compared to homogenates of beef carcass. As co-incubation of O157 with background microbiota complex from bovine intestinal products in buffered peptone water, in the absence of meat samples, tended to reduce the extent of growth of O157, we reasoned that certain microbiota present in offal products played a role. In support of this, inoculation of generic E. coli from bovine intestinal products into the homogenates significantly reduced the extent of growth of O157 in the homogenates of bovine intestinal and loin-beef products, and this effect was markedly increased when these homogenates were heat-treated prior to inoculation. Together, this report provides first evidence of the prevalence of STEC in a variety of bovine offal products in Japan. The prevalence data herein may be useful for risk assessment of those products as a potential source of human STEC infection beyond the epidemiological background. The growth characteristic of STEC O157 in offal products also indicates the importance of being aware when to test these food products.

Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta).

The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as "nori", is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of "iroochi", a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.

The occurrence of various collagen diseases in one family: a sister with ISSc, PBC, APS, and SS and a brother with systemic lupus erythematosus.

We encountered siblings who had collagen diseases and related symptoms. Case 1 was a 53-year-old woman who had limited cutaneous systemic sclerosis (ISSc) associated with primary biliary cirrhosis (PBC), antiphospholipid antibody syndrome (APS), and subclinical Sjögren's syndrome (SS). Case 2 was a 48-year-old man, her younger brother, with systemic lupus erythematosus (SLE) that developed at 32 years of age. Investigation of their family revealed that their mother had Raynaud's phenomenon, arthritis, and subclinical Sjögren's syndrome, and that another younger brother of Cases 1 and 2 had Raynaud's phenomenon and general fatigue. HLA analysis revealed that the sister and brother had some identical HLA antigens in common, including A2, A33 (19), B67, B44 (12), Cw7, DR2, DR6, DR52, and DQ1. The sister, brother and their mother had common HLA antigens including A2, B67, Cw7, DR2, and DQ1. Although Cases 1 and 2 shared the same HLA system, they presented different phenotypes of collagen disease.

New gamma-fluoromethotrexates modified in the pteridine ring: synthesis and in vitro immunosuppressive activity.

Our continuing program to develop new antifolate drugs useful against rheumatoid arthritis led us to modify the pteridine ring of gamma-fluoromethotrexate. Pyrrolopyrimidine derivatives 1e and 1t were found to exhibit potent suppressive effects on the responses of both T and B cells to mitogens, although tetrahydropyridopyrimidine derivatives 2e and 2t and quinazoline derivatives 3e, 3t and 4e showed very weak suppressive activities. Thus, conversion of the pteridine ring of gamma-fluoromethotrexate to a pyrrolopyrimidine ring led to a new potential antirheumatic compound.

Differential scanning calorimetry of light meromyosin fragments having various lengths of carp fast skeletal muscle isoforms.

Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10 degrees C and 30 degrees C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10 degrees C- and 30 degrees C-acclimated carp, respectively. These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525. Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10 degrees C and 30 degrees C types in our expression system using Escherichia coli. All the LMM fragments except for the 10- and 30-2/4 quarters for the 10 degrees C and 30 degrees C types, respectively, exhibited a typical pattern of a-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30 degrees C-type LMM fragments were all found to be more thermostable than the 10 degrees C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10 degrees C- and 30 degrees C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses. For three mutants in which two residues of the 10 degrees C type were replaced by those of the 30 degrees C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/H536Q, the endothermic peaks in DSC increased by 1.4-2.0 degrees C from that of the original 10 degrees C type. The T(m) values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3 degrees C higher than that for the 10 degrees C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in T(m) values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6 degrees C, which roughly corresponds to that between the original 10 degrees C and 30 degrees C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10 degrees C- and 30 degrees C-type LMMs.

Two proline-rich nuclear localization signals in the amino- and carboxyl-terminal regions of the Borna disease virus phosphoprotein.

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids.

A novel Rab GTPase, Rab33B, is ubiquitously expressed and localized to the medial Golgi cisternae.

Small GTP-binding proteins of the Rab family play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. In mammals, more than 30 proteins belonging to the Rab family have been reported to date. We report here the molecular cloning and characterization of a novel Rab protein, Rab33B. The amino acid sequence of Rab33B shows 55.3% identity to the Rab33A protein (previously called S10), and these two proteins share unique amino acid sequences at the effector domain. The genomic organization of rab33B was the same as rab33A: it consists of two exons. Thus, these two proteins make a subclass within the Rab family. Northern blot analysis showed that rab33B is expressed ubiquitously in mouse tissues, in contrast to rab33A whose expression is restricted to the brain and the immune system. A 26 kDa protein was detected by western blotting using a Rab33B-specific monoclonal antibody. Using immunofluorescence studies, Rab33B was shown to co-localize with (alpha)-mannosidase II, a Golgi-specific marker. Immunoelectron microscopy analysis further defined the localization of Rab33B to the medial Golgi cisternae. These results suggest Rab33B plays a role in intra-Golgi transport.

Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs.

Differential scanning calorimetry (DSC) was performed to investigate thermodynamic properties of three carp fast skeletal light meromyosin (LMM) isoforms expressed in Escherichia coli by recombinant DNAs. Three isoforms were the 10 degreesC-, intermediate-, and 30 degreesC-type LMM predominantly expressed in carp acclimated to 10, 20, and 30 degreesC. The isoforms expressed in E. coli by recombinant DNAs exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm. Moreover, the three isoforms formed paracrystals typical of LMM, suggesting that expressed proteins retained intact structural properties. When the LMM isoforms were subjected to DSC analysis, the 10 degreesC and 30 degreesC types showed endotherms having transition temperatures (Tm) at 35.1 and 39.5 degreesC, respectively, which are responsible for thermal unfolding of alpha-helix. The intermediate type exhibited two comparable endotherms with Tm values at 34.9 and 40.6 degreesC, implying that it has intermediate thermodynamic properties between those of 10 degreesC and 30 degreesC types. However, a chimeric LMM having the 10 degreesC and 30 degreesC type as N- and C-terminal halves, respectively, showed the DSC pattern typical of the whole 30 degreesC-type molecule. On the other hand, another chimeric LMM composed of the N-terminal 30 degreesC type and C-terminal 10 degreesC type gave the pattern of the full 10 degreesC type. These results suggest that thermodynamic properties of the C-terminal half largely account for thermal unfolding of the whole molecule.

Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein.

The Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants lacking the 13 NH2-terminal amino acid residues 1MPPKRRLVDDADA13 in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of 4KRR6 by 4NSG6 was retained in the cytoplasm. Furthermore, a nonapeptide, 3PKRRLVDDA11, derived from the NH2-terminal region of N conferred nuclear targeting activity to beta-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where 4KRR6 basic amino acid residues are located.

Interaction of pigeon cytochrome c-(43-58) peptide analogs with either T cell antigen receptor or I-Ab molecule.

We determined that a pigeon cytochrome c-derived peptide, p43-58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43-58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43-58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vbeta usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR beta chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR beta chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

Differential scanning calorimetry and CD spectrometry of acclimation temperature-associated types of carp light meromyosin.

Differential scanning calorimetry and CD spectrometry were employed to study the thermal unfolding of light meromyosin (LMM) prepared from carp acclimated to different temperatures. The transition temperatures given by the major peaks at pH 8.0 in 0.6 M KCl for LMM from carp acclimated to 10 degrees C were 32.5 and 39.5 degrees C with the calorimetric enthalpies (DeltaHcal) of 269 and 52 kcal/mol, respectively. LMM from carp acclimated to 20 degrees C exhibited three peaks of transition temperatures at 34.5, 40.2, and 46.9 with DeltaHcal of 152, 20, and 10 kcal/mol, respectively. On the other hand, LMM from carp acclimated to 30 degrees C showed two different patterns. The first experiment gave two transition temperatures at 39.2 and 47.3 degrees C with DeltaHcal of 231 and 39 kcal/mol, respectively. The second series of experiments resulted in showing three peaks of 34.4, 39.5, and 47.5 degrees C with DeltaHcal of 117, 123, and 28 kcal/mol, respectively. N-terminal amino acid sequence analysis revealed that LMM at the second series of experiments with the 30 degrees C-acclimated carp contained component(s) predominant in the 20 degrees C-acclimated carp. Thermal unfolding responsible for these transition temperatures was well explained by melting of alpha-helices which could be determined by far-ultraviolet CD spectroscopy. These results clearly demonstrate that the 30 degrees C-acclimated carp contained the most thermostable LMM.

Genomic organization of the mouse adrenocorticotropin receptor.

As a step toward understanding the transcriptional regulation of the adrenocorticotropin receptor (ACTH-R) gene, we examined the full length cDNA sequence of the mouse ACTH-R by rapid amplification of cDNA ends, and the organization of the gene. Mouse ACTH-R mRNA consists of 374 bp in the 5'-untranslated region (UTR), 888 bp in the coding sequence, and 445 bp in the 3'-UTR, the 1707 bp being fairly compatible with the 1.8-kb adrenal mRNA detected by Northern analysis. The mouse ACTH-R gene consists of at least four exons; the first three exons encode 5'-UTR and the fourth exon encodes part of 5'-UTR, the entire coding region, and the whole of 3'-UTR. We also defined two mRNA species, one with and one without the 57-bp exon 2, produced by alternative splicing.

Structure and expression of the mouse S10 gene.

We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human S10. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain.

Amplification of a full-length Borna disease virus (BDV) cDNA from total RNA of cells persistently infected with BDV.

We have developed a novel reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.

cDNA cloning of myosin heavy chain isoforms from carp fast skeletal muscle and their gene expression associated with temperature acclimation.

We have isolated cDNA clones encoding fast skeletal muscle myosin heavy chains of carp acclimated to 10, 20 and 30 degrees C for over 5 weeks. All clones covered at least the full length of L-meromyosin, the C-terminal part of the myosin molecule. Nucleotide sequence analysis on cDNA clones showed three types of 3' untranslated sequences, demonstrating that carp expresses at least three myosin heavy chain isoforms in fast skeletal muscle in an acclimation-temperature-dependent manner. cDNAs were identified which were the predominant types expressed in 10 degrees C- and 30 degrees C-acclimated fish, as well as an intermediate type present at all acclimation temperatures. Northern blot analysis using probes of three kinds of DNA fragments from the 3' untranslated region of carp acclimated to 10, 20 and 30 degrees C further confirmed the presence of acclimation-temperature-specific isoforms. In addition, it was found that mRNA levels of three isoforms were altered in an acclimation-temperature-dependent manner. When the deduced amino acid sequences of three types of carp L-meromyosin were compared with those of homoiotherms, the 30 degrees C-acclimated type was more similar to those of homoiotherms than was the 10 degrees C-acclimated type.

A novel missense mutation in the thyroid hormone receptor beta gene in a kindred with resistance to thyroid hormone.

Direct sequencing of exon 9 of the thyroid hormone receptor beta (TRbeta) gene in a kindred with resistance to thyroid hormone revealed a substitution of threonine for methionine in codon 313 in one allele resulting from a T to C transition. This is a novel missense mutation that resides in one of the two mutational "hot-spot" regions of the TR beta gene suggesting altered triiodothyronine binding to this mutant receptor.