Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections.

Non-isotopic in situ hybridization with a novel phenytoin (PHE)-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-11(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 microgram/ml), and then heated at 70 degrees C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37 degrees C overnight. The hybridization signal was visualized by alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate (BCIP)/4-nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections.

Development of a supersensitive polymerase chain reaction method for human T lymphotropic virus type II (HTLV-II) and detection of HTLV-II proviral DNA from blood donors in Japan.

A supersensitive polymerase chain reaction procedure was developed to detect human T-lymphotropic virus type II (HTLV-II) proviral genome. Six primer pairs covering the various regions of HTLV-II were compared and selected on the basis of specificity and sensitivity. Among them, one primer pair of the pol region of HTLV-II (II pol) was able to amplify and detect even 0.1 fg of the cloned plasmid HTLV-II DNA (seven copies) by regular ethidium bromide staining on polyacrylamide gel. By using this procedure, we screened 189 HTLV-I seropositive blood donors from Yamaguchi and Fukuoka Red Cross Blood Centers, Japan. There were four positive samples detectable with the HTLV-II-specific pol primer pair, as well as with the HTLV-I tax primer pair. The amplified DNAs of two specimens were cloned and sequenced. The sequences of the HTLV-I tax region from both specimens were identical to that of HTLV-I. On the other hand, those of the HTLV-II pol region were identical to that of HTLV-II, except for one base substitution in a clone from one subject. These results indicate that dual infection of HTLV-I and HTLV-II in the same persons occurs among Japanese blood donors.

Synthesis of proteins in Escherichia coli immunoreactive with sera from individuals infected with human T-cell leukemia virus type I.

The env-pX IV fused gene of human T-cell leukemia virus type I (HTLV-I) was inserted into lac promoter-directed expression vectors for production of viral proteins in bacteria. Resulting recombinant plasmids, pK13 and pK15, directed synthesis of fused proteins of 59 kDa (Env-p40x) and 100 kDa (Gag-Env-p40x), respectively. Western blot analysis showed that these proteins were reactive with sera of patients with adult T-cell leukemia (ATL) and retained multiple antigenic determinants of viral proteins. In combination with recombinant Gag protein [S. Itamura, K. Shigesada, M. Imai, N. Kobayashi, T. Hamakado, T. Harada and M. Hatanaka, Gene 38, 57-64 (1985)], these bacterially synthesized proteins may provide a useful tool for differential diagnosis of ATL by detecting serum antibodies against individual viral proteins and for analysis of viral gene functions.

Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use.

An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene. Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx. 0.3% of total soluble proteins. The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography.

Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by Actinomadura sp.

A new inhibitor of angiotensin I converting enzyme, I5B2, was isolated from the culture broth of Actinomadura sp. No. 937ZE-1. This compound contains N-methylvaline, tyrosine and 1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid. The microorganism also produced another inhibitor, I5B1, which is identical with K-4 isolated from Actinomadura sp. as an antihypertensive agent.

Isolation and characterization of ancovenin, a new inhibitor of angiotensin I converting enzyme, produced by actinomycetes.

Ancovenin, an inhibitor of angiotensin I converting enzyme isolated from the culture broth of a Streptomyces species, is a dialysable peptide composed of sixteen amino acid residues containing unusual amino acids such as threo-beta-methyllanthionine, meso-lanthionine, and dehydroalanine.