Evaluation of the drug lymphocyte stimulation test (DLST) with shosaikoto]

BACKGROUND: Our aim is to evaluate the significance of DLST by Shosaikoto. METHODS: We clinically evaluated 3 cases of drug-induced pneumonia assumed to be caused by Shosaikoto, and we performed DLST of Shosaikoto for healthy controls, and compared the data with drug-induced pneumonia cases of Shosaikoto. RESULTS: As clinical characteristics of 3 cases, 2 cases were positive for hepatitis C virus antibody, and 1 case was positive for DLST of Shosaikoto. The observed chest high-resolution CT (HRCT) findings showed hypersensitivity pneumonia (HP) pattern in all 3 cases. Prognosis was good in all 3 cases. DLST of Shosaikoto was positive in 27.5% of healthy controls. Stimulation index (S.I.) of DLST in drug-induced pneumonia cases increased depending on drug dilution density, compared to that of healthy controls. CONCLUSION: DLST of Shosaikoto showed high false-positive rate. However, we may be able to distinguish the true-positive cases with the false-positive cases by comparing the S.I. of DLST according to drug dilution density.     

Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.     

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.     

Syntheses of polymeric cobalt dinitrogen complexes and reduction of the coordinated nitrogen

Polymeric cobalt dinitrogen complexes were synthesized by two different methods: by the ligand substitution reaction of polymers 5a – c and 6a – c , containing 4-diphenylphosphinophenylethylene units ( 3 ) or 4-diphenylphosphinomethylphenylethylene units ( 4 ), respectively, with hydridodinitrogentris(triphenylphosphine)cobalt(I) (CoH(N₂)(PPh₃)₃), or by the direct reaction of tris(acetylacetonato)cobalt(III) with triphenylphosphine, the polymers 5a – c or 6a – c , and triisobutylaluminium under nitrogen. The intensity of the v band in the IR spectrum of the polymeric dinitrogen complex was found to be about four times as that of CoH(N₂)(PPh₃)₃. Ammonia was formed after hydrolysis of the complex, which was prepared by mixing a tetrahydrofuran solution of naphthaline, lithium, and titanium tetrachloride with the polymeric dinitrogen complex. The yields of ammonia in the case of the polymeric complexes were high in comparison with that obtained with CoH(N₂)(PPh₃)₃. The highest yields of ammonia were obtained when a mole ratio [P?]/[Co] < 1 ([P?] is the concentration of phosphino groups in the polymer and [Co] is that of Co in CoH(N₂)(PPh₃)₃), a temperature of ?20°C, and a reaction time of 1 h were applied.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

De novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)] with intact blood group-MN locus,confining its locus to 4q28.2–4q31.1

We report a malformed female infant withde novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)]. The MN blood type analysis of the family members showed that the patient had an intact blood group-MN locus. The locus of the gene responsible for the MN antigen activity is confined to a 4q28.2–4q31.1 segment on the basis of the result of this patient and the previous mapping data.     

New genome type of adenovirus serotype 19 causing nosocomial infections of epidemic keratoconjunctivitis in Japan

Twelve strains of adenovirus serotype 19, isolated from cases of epidemic keratoconjunctivitis in Japan in 1992, 1993, 1997, and 1998, were analyzed by DNA restriction analysis, using restriction endonucleases BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, SacI, SalI, SmaI, and XhoI. Among these 11 restriction endonucleases, EcoRI, PstI, SacI, and SmaI were discriminative enzymes, showing restriction patterns different from those reported previously for the prototype and the variant 19a. This new genome type was isolated in 1997 and 1998, when an increase of epidemic keratoconjunctivitis cases caused by adenovirus serotype 19 was observed for both sporadic and nosocomial infections. Strains from 1992 and 1993 showed restriction patterns similar to those of the worldwide reported variant 19a for all enzymes used. The changes detected in strains from 1997 and 1998 could be the reason for the recent epidemic.     

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).     

Genetic mapping of genes regulating the thymus size in back-cross rats between the laboratory BUF/Mna strain and the MITE strain derived from the wild rat, Rattus norvegicus

The thymoma prone BUF/Mna (B) rat is a useful model for Studying the genes responsible for thymus enlargement during the stage of young growth. Among the strains of rats, B rats have the largest thymuses at al stages of life. A locus, Ten-1 , which contributes to thymus enlargement in back-cross (BC) rats between the B and WKY/NCrj (W) strains, was mapped on chromosome 1. To determine the precise location of the bus, (B×(B×MITE)F1) BC rats were generated by crossing the B strain with the Inbred MITE (M) strain, which was established from captured, Japanese wild rats, and were examined by linkage study using polymerase chain reaction with 67 microsatellite markers. Linkages with thymus enlargements were found In genotypes of seven markers, BSIS, LSN, MYL2, IGF2, PBPC2, D1Mgh11 , and D1Mit6 , by X^2-test and Student's t -test, which confirmed the presence of the genetic locus associated with thymus enlargement, Ten-1 , in this region. Paradoxically, a suppressive locus, Tsu-1 , to thymus enlargement was also found on chromosome 3, showing linkages of phenotype of the small thymus with genotypes of SCN2A, CAT D3Mit16 , and D3Mit13 . By analyses of mapmaker/exp and mapmaker/qtl, Ten-1 was mapped at 4.6 cM proximal from IGF2 locus on chromosome 1 and Tsu-1 at 4.0 cM proximal from CAT locus on chromosome 3, respectively.