Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

Mouse interferons: amino terminal amino acid sequences of various species.

Mouse interferons of three size classes (A, 35,000 to 40,000 daltons; B, 26,000 to 33,000 daltons; and C, 20,000 daltons) were purified from Ehrlich ascites tumor cells infected with Newcastle disease virus. The sequences of the first 24 amino acids (No. 17 has not been identified) of interferons A and B are identical. The sequence of the first 20 amino acids of interferon C differs from that of A and B in 18 positions. There is partial homology in amino terminal sequence between mouse interferons A (or B) and a human fibroblast interferon and between mouse interferon C and a human lymphoblastoid interferon.

Effect of interferon-alpha 1 from E. coli on some cell functions.

Interferon-alpha 1 from Escherichia coli transformed with a hybrid plasmid containing a human leukocyte complementary DNA insert, induces resistance to virus in appropriate target cells. It also shares the following properties with natural leukocyte interferon (IFN). (i) It enhances natural killing activity of human lymphocytes, (ii) it enhances antibody-dependent cell-mediated cytotoxicity, (iii) it suppresses antigen- and mitogen-induced leukocyte migration inhibition, (iv) it inhibits growth of IFN-sensitive Burkitt lymphoma cells. Since these activities are exhibited by a cloned protein species, they are due to IFN itself and not to other human proteins.

Activity of three-beta-hydroxysteroid dehydrogenase in granulosa cells treated in vitro with luteinizing hormone, follicle-stimulating hormone, prolactin, or a combination.

Three-beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the pathway that produces progesterone. Hy-Line hens (W36, W98, and Brown) were subjected to mild heat stress [36 degrees C for 24 h (acute heat stress, AHS) or 2 wk (chronic heat stress, CHS)] or maintained at 22 degrees C (thermoneutral, TN). Granulosa cells (GC) from the 3 largest follicles were isolated, dispersed, and incubated with luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), a combination, or no hormone (control), and then with pregnenolone nitro blue tetrazolium to determine 3beta-HSD activity. Treatment by LH (TN, P = 0.04; AHS, CHS, P < 0.0001) and by LH+FSH (TN, AHS, CHS, P < 0.0001) resulted in increased enzyme activity compared with the respective controls. In TN and CHS, LH+FSH increased the activity to a greater extent than LH alone (TN, P = 0.02; CHS, P = 0.0004); in AHS the increase was not significant (P = 0.29). Treatment with FSH, PRL, or LH+PRL decreased (TN, AHS) or had no effect (CHS) on 3beta-HSD activity. In TN and AHS cells, FSH and PRL reduced enzyme activity (P = 0.006 and 0.0580, respectively). When LH was added to PRL, suppression by PRL was mitigated somewhat. When LH and FSH were added to PRL, 3beta-HSD activity in AHS and CHS cells actually increased compared with the respective controls (P = 0.052 and 0.003) but remained below the activity of cells incubated with LH+FSH or LH alone. This suggests that gonadotropic actions of LH and LH+FSH are countered by the antigonadotropic action of PRL and, conversely, that PRL reduces the stimulatory action of LH and FSH. Strain differences in GC response to hormones were observed primarily in the CHS-treated birds; generally, W98 was highest; Browns showed the weakest response, and W36 was intermediate. In earlier studies, HS reduced circulating LH and GC progesterone and 3beta-HSD activity in vitro and increased circulating PRL. The results suggest a mechanism by which reduced activity of 3beta-HSD and progesterone by GC during HS might be explained, particularly with the differences in strains observed.

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced my human leukocytes and by E. coli.

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.

Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E. coli cells and eucaryotic cells.

To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC-F, was constructed by inserting cDNA for the F protein into a pUC vector. Upon induction of E. coli cells transformed with pUC-F, a new protein was obtained, which was identified as Fo on Western blot analysis. The cDNA fragment for the F gene was excised from pUC-F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL-F. COS-1 cells transfected with pSVL-F gave a band on SDS-gel electrophoresis which corresponded to the size of the Fo proteins.

Cloning and sequencing of the cDNA encoding rice elongation factor 1 beta'.

A cDNA clone coding for rice elongation factor 1 beta' (EF-1 beta') was isolated from a rice anther cDNA library. The clone, named RB', was 980 bp long and contained a single open reading frame coding for 223 amino acids; the first 31 amino acids, except for the first methionine, which is absent in the mature protein, are identical to those of the purified protein determined with a protein sequencer. The amino acid sequence of rice EF-1 beta' shows homology to the C-terminal half of Artemia salina EF-1 beta (59%) and human EF-1 beta (63%), but might not have a phosphorylation site for casein kinase II which has been conserved in Artemia saline EF-1 beta and EF-1 delta, human EF-1 beta and silkworm EF-1 beta'.

Cloning and characterization of the cDNA encoding rice elongation factor 1 beta.

We have cloned and sequenced a cDNA coding for rice elongation factor 1 beta (EF-1 beta). The clone was 1420 bp long and contained an open reading frame coding for 229 amino acids. The overall identity between rice EF-1 beta and rice EF-1 beta' [Matsumoto, S., Oizumi, N., Taira, H. and Ejiri, S. (1992) FEBS Lett. 311, 46-48] is 60% at the amino acid sequence level; a higher percent identical residues (81%) were especially observed in the C-terminal region. Rice EF-1 beta has no conserved phosphorylation site for casein kinase II and no leucine zipper motif, although these motifs are well conserved in EF-1 delta (= beta in plants) subunits of animal EF-1.

Angiotensin I-converting enzyme in human placenta.

Angiotensin I-converting enzyme (ACE, peptidyldipeptide hydrolase, kininase II, EC 3.4.15.I) from human placenta was purified 6297-fold and characterized. ACE could be extensively purified by affinity chromatography with Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), an orally active antihypertensive agent and a potent inhibitor of this enzyme. Its molecular weight and subunit size were estimated to be 300 000 by high-performance gel permeation chromatography and 85 000 by sodium dodecyl sulphate gel electrophoresis, respectively, indicating its polymeric structure.

Kinetics of interactions of sendai virus envelope glycoproteins, F and HN, with endoplasmic reticulum-resident molecular chaperones, BiP, calnexin, and calreticulin.

Sendai virus envelope glycoproteins, F and HN, mature during their transport through the endoplasmic reticulum (ER) and Golgi complex. To better understand their maturation processes in the ER, we investigated the time course of their interactions with three ER- resident molecular chaperones, BiP, calnexin (CNX), and calreticulin (CRT), in Sendai virus-infected HeLa cells. Pulse-chase and immunoprecipitation analyses using antibodies against each virus glycoprotein or ER chaperone revealed that F precursor interacted with CNX transiently (t(1/2)=8 min), while HN protein displayed longer and sequential interactions with BiP (t(1/2)=8 min), CNX (t(1/2)=15 min), and CRT (t(1/2)=20 min). HN interacted with the three ER chaperones not only as a monomer but also as a tetramer for several hours, suggesting mechanism(s) to undergo chaperone-mediated quality control of an assembled HN oligomer in the ER. The kinetics of dissociation of the HN-chaperone complexes exhibited a marked delay in the presence of proteasome inhibitors, suggesting that a part of HN associated with BiP, CNX, and CRT is destined to be degraded in the proteasome-dependent pathway. Further, the associations between virus glycoproteins and CNX or CRT were impaired by castanospermine, an inhibitor of ER glucosidase I and II, confirming that these interactions require monoglucosylated oligosaccharide on F(0) and HN peptides. These findings together suggest that newly synthesized F protein undergoes rapid maturation in the ER through a transient interaction with CNX, whereas HN protein requires more complex processes involving prolonged association with BiP, CNX, and CRT for its quality control in the ER.

The target reaction in the antiviral action of mouse interferon against vesicular stomatitis virus multiplication.

Treatment of mouse L929 cells with mouse interferon (IFN) lowered the yield of vesicular stomatitis virus (VSV) in a dose-dependent manner. Accumulation of viral proteins was severely inhibited in IFN-treated cells, whereas cellular protein synthesis was not, indicating that the virus-induced shutoff of cellular protein synthesis was prevented by IFN. In order to identify the major target of IFN action precisely, the effect of IFN treatment on the synthesis of viral RNAs and proteins at various stages during the course of viral replication was examined. Accumulation of viral RNAs late in infection was inhibited, as was the case with viral proteins, but the synthesis of leader RNA and mRNAs early in infection was not significantly inhibited by treatment with a moderate dose of IFN. On the other hand, viral protein synthesis at an early stage of infection was strongly inhibited by IFN. The results indicate that the major target reaction of antiviral action of IFN against VSV multiplication is the translation of viral mRNA.

The roles of individual cysteine residues of Sendai virus fusion protein in intracellular transport.

The role of intramolecular disulfide bonds in the fusion (F) protein of Sendai virus was studied. The 10 cysteine residues were changed to serine residues using site-directed mutagenesis. None of the cysteine mutant F proteins reacted with a monoclonal antibody specific for the mature conformation of the F protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. The transport of these mutant proteins to the cell surface was drastically reduced. All of the cysteine mutant F proteins remained sensitive to endoglycosidase H (endo H) for 3 h after their synthesis. Moreover, cell surface transport of the hemagglutinin-neuraminidase (HN) protein co-expressed with each of these cysteine mutant F proteins was also reduced. These results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the F protein, and that interaction of the F and HN proteins takes place intracellulary.

Synthesis and biological activities of beta-alanyltyrosine derivative of 2',5'-oligoadenylate, and its use in radiobinding assay for 2',5-oligoadenylate.

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.

Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.

Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates.

The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2. The results suggest that the P1 reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein was used as a partner Gln-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.

Properties of tofus and soy milks prepared from soybeans having different subunits of glycinin.

The contribution of soybean protein to the physical properties of tofu, a product manufactured by curdling soy milk with coagulants such as calcium or magnesium chloride, was studied by comparing the properties of soy milk prepared from soybeans with different subunits (I, IIa, and IIb) of glycinin with amino acid residues deleted. The breaking stress value of the tofu curds prepared from soybeans having group I was higher than those without group I. The soy milks having group I contained more protein particles and showed more sensitivity to calcium and magnesium ions than those without group I. The amounts of glycinin and protein particles were higher in the soy milks having group I than those in the soy milks without group I. To elucidate the influence of each group on the breaking stress, the glycinin content was adjusted to an identical level in soy milks having each group. Among the tofu curds from three groups, their order of hardness according to their breaking stress was IIa, IIb, and I. The order of particle content among these soy milks was also IIa, IIb, and I. Therefore, the results suggested that the breaking stress value of the tofu curd is dependent upon the number of protein particles in the soy milk and that the number of the particles is determined by the proportion and structure of glycinin in the soybean.

3M integral bipolar cup system for dysplastic osteoarthritis. Clinical and radiographic review with five- to seven-year follow-up.

Between 1995 and 1997 we undertook 40 bipolar hip arthroplasties in 35 patients with dysplastic osteoarthritis. The steep and shallow acetabulum was excavated and the bipolar socket was placed high with an adjustment of leg-length. At follow-up of between five and seven years, there were 19 excellent, 16 good and five fair results according to the scoring system of Merle d'Aubigné and Postel. The mean radiographic superior migration of the bipolar socket was 2.1 mm (0 to 10). Osteolysis was noted in three hips within three years of the operation. Abduction on weight-bearing was recorded in 24 hips and the bipolar system was found to be functioning predominantly between the inner bearing and the metal femoral head in 20.

Spread of swine hemagglutinating encephalomyelitis virus from peripheral nerves to the CNS.

Swine hemagglutinating encephalomyelitis virus (HEV) strain 67N was inoculated into the sciatic nerve or the right leg crural muscle of rats. In both cases, the virus was isolated first from the caudal half of the spinal cord on day 2 after inoculation, and from the rostral half of the spinal cord and the brain on day 3. The virus titers in the brain reached a maximum when the infected rats developed CNS symptoms on day 5. Using confocal laser scanning microscope, fluorescent positive cells were first found in the lumbar dorsal root ganglion (DRG) and spinal cord ipsilateral of the inoculated leg on day 3. Antigen positive neurons were found bilaterally in the lumbar DRG and spinal cord on day 4. On day 5 specific fluorescence was observed in the neurons of the cerebral cortex, hippocampus, brainstem and Purkinje cells in the cerebellum.

Correlation between renal blood flow and intrarenal Doppler measurements in canine autotransplanted kidney.

For proper interpretation of the changes in intrarenal Doppler ultrasound measurements, we evaluated the direct correlation between total renal blood flow and intrarenal Doppler parameters. Under progressive constriction of the renal artery in canine autotransplanted kidneys. we simultaneously measured blood flow at the main renal artery and Doppler parameters at the segmental artery. The changes in total renal blood flow were well correlated to changes in peak systolic velocity, end diastolic velocity and resistive index (RI) of the segmental artery (r = 0.964, 0.960 and 0.486, respectively). The acute reduction of total renal blood flow produces a linear decrease in Doppler parameters at intrarenal arteries. These results should be helpful for better understanding the changes in renal hemodynamics in various pathologic conditions as well as those induced by various vasoactive agents including angiotensin converting enzyme inhibitor.

[Relationships between atmospheric Sugi (Japanese cedar)-pollen counts and indices of climatic conditions].

Atmospheric pollen surveys have been conducted using Durham's standard sampler for 12 years in Toyama Prefecture. The relationships between Sugi (Japanese cedar, Cryptomeria japonica D. Don) pollen and some indices of climatic conditions were analyzed. The results were as follows: 1. Total pollen counts showed a tendency to increase for 12 years, although the variation of the count was relatively large. 2. Significant correlations were found between pollen counts and some indices of the climatic conditions in July of the previous year, such as duration of sunshine, mean daily temperature and daily maximum, as well as minimum temperature. 3. Significant correlation was found between pollen count and duration of sunshine in January and March, and the amount of snowfall in March of the observed year using multiple regression analysis. Pollen counts were shown to be relatively high in years with short sunshine in January and long sunshine and heavy snow in March.