Radioimmunoassay of chenodeoxycholic acid-3-sulfate in urine

A sensitive and specific radioimmunoassay (RIA) for the measurement of urinary total chenodeoxycholic acid-3-sulfate (SCDCA) was developed and the accuracy was confirmed. SCDCA bound to bovine serum albumin as the antigen and emulsified with Freund's complete adjuvant was injected into rabbits. The antiserum obtained was capable of binding 75% of [11,12-3H]SCDCA at 1:1000 dilution. The percentage of bound radioactivity decreased linearly with logarithmic increases in unlabeled SCDCA, from 8 to 200 pmol/ml. The antiserum showed an extremely high specificity for SCDCA (free and conjugated), and the values determined by RIA indicated a close correlation with those found by gas-liquid chromatography. The daily urinary SCDCA level was determined using SCDCA-RIA in 12 disease-free humans and 74 patients with chronic liver diseases. In the normal subjects the daily urinary SCDCA level was 0.74 +/- 0.83 mg/day and increased levels were evident in all groups with chronic liver diseases. The daily urinary SCDCA level corresponds closely with the extent of hepatic dysfunction.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Matrix Gla protein C-terminal region binds to vitronectin. Co-localization suggests binding occurs during tissue development.

Matrix Gla protein (MGP) regulates calcification in cartilage and arteries. MGP synthesis during embryonic development and its binding and regulation of growth factors and morphogens of the TGF-beta/BMP superfamily suggests that it has additional functions. Assay by far-western gel overlays and gel filtration shift shows MGP binds vitronectin. Binding is saturable and consistent with a single class of binding sites. MGP binds to vitronectin but not collagen, fibromodulin, heparin, osteocalcin, chondroitin sulfate, laminin, ovalbumin or albumin. We have identified a vitronectin binding site within a 17-amino acid peptide 61-77 near the carboxyl-terminus that corresponds to a naturally occurring MGP C-terminus. MGP and the 61-77 MGP peptide also binds to fibronectin. MGP and vitronectin are focally co-localized in embryonic tissues. Co-localization in vivo suggests that the MGP and vitronectin interactions may modify cell-matrix interactions. Alternatively, vitronectin-bound MGP may have altered function for modulating BMP2 or TGF-beta activity. The current study demonstrates that MGP has a novel binding activity for vitronectin, an extracellular protein that promotes cell-matrix interactions and regulates coagulation.     

Nucleotide sequence of the PR-1 gene of Nicotiana tabacum

A gene encoding one of the pathogenesis-related proteins, PR1a, and two related pseudogenes were isolated from Nicotiana tabacum. The cloned PR1a gene (pPR-gamma) and one of the pseudogenes (pPR-alpha) were sequenced and found to have similar structures. The sequence of pPR-gamma was quite similar to that of the cDNA clone of PR1a. The plasmid pPR-gamma did not contain an intron and had a typical promoter sequence in the 5'-flanking region.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

Pharmacological intervention in oxidant-induced calcium pump dysfunction of dog heart

Micromolar concentrations of HOCl, an oxidant produced by activated neutrophils, inhibited Ca2+ uptake and Ca2+ATPase of isolated dog heart sarcoplasmic reticulum (SR). DTT antagonized completely the HOCl effect only when it was given within 5 min after the addition of HOCl. When the pharmacological intervention was delayed, the recovery with DTT was not complete, and administration of DTT 30 min after the start of HOCl's reaction with SR resulted in only a small improvement in SR Ca2+ uptake. Although H2O2 and Fe ion-chelate (a free radical-generating procedure) also inhibited Ca2+ uptake and ATPase, the concentrations required were very large. The response of cardiac sarcolemmal and skeletal muscle SR calcium pumps to oxidants was similar to that of the cardiac SR calcium pump.     

Localization of a determinant mediating partial macrolide resistance in Staphylococcus aureus

Four out of more than 8,200 Staphylococcus aureus strains isolated in Japan between 1961 and 1980 were constitutively resistant to a variety of macrolide antibiotics except tylosin and rokitamycin, but susceptible to lincosamide and streptogramin type B antibiotics (PM). The data obtained by agarose gel electrophoresis, CsCl-ethidium bromide density gradient analysis, diagnosis with ATP-dependent deoxyribonuclease, and a test transducing into a rec- mutant with phage 80L2 propagated on PM-resistant S. aureus all suggested that the determinant for the PM-resistance is located in chromosome.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.