Hisanaga Igarashi

Physical and Functional Interactions of Doc2 and Munc13 in Ca2+-dependent Exocytotic Machinery

Doc2 has two C2 domains that interact with Ca2+ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca2+-dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K+-induced Ca2+-dependent exocytosis. The Doc2-Munc13 interactions are stimulated by phorbol ester through the C1 domain of Munc13. Overexpression of the Doc2-interacting domain of Munc13 reduces the Ca2+-dependent exocytosis from PC12 cells, and co-expression with Doc2 suppresses this reduction. These results, together with the earlier findings that secretagogues produce DAG and elevate cytoplasmic Ca2+, suggest that the DAG-induced Doc2-Munc13 interactions play an important role in Ca2+-dependent exocytotic machinery.

Cloning, characterization and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factor

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N‐terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S‐transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.

Serologic Evidence That Streptococcal Superantigens Are Not Involved in the Pathogenesis of Kawasaki Disease

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti‐superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin‐producing streptococcal infection and the onset of KD.

A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein

The pX sequence of human T cell leukemia virus type 1 (HTLV‐1) has been thought to be expressed as a doubly spliced mRNA that codes for p40tax, p27rex and p21X. However, we identified a novel alternatively spliced mRNA in the HTLV‐1 infected cells by using reverse transcription followed by the polymerase chain reaction. This mRNA contains only the first and third exons of the doubly spliced mRNA and encodes only p21X. Our data that this mRNA is responsible for expressing p21X exists in most of HTLV‐1 infected cells strongly suggests that p21X may play a crucial role for HTLV‐1 replication.

Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat

We have identified and analyzed a 27‐nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T‐cell leukemia virus type I (HTLV‐I) long terminal repeat (LTR) which is required for HTLV‐I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T‐cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2‐to 5‐fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (USRE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with silver staining: 110‐, 80‐ and 70‐kDa proteins. The 110‐kDa protein appeared to be a novel DNA‐binding protein whose characteristics are still obscure, while the 70‐ and 80‐kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV‐I basal transcriptional repression.

Molecular cloning of mouse Doc2α and distribution of its mRNA in adult mouse brain

We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2alpha. Doc2alpha is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2(+)-dependent exocytosis. We have isolated here a mouse Doc2alpha cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2alpha cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2alpha is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2alpha is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2alpha is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2alpha plays a specific role in the area where it is expressed.

Human T cell leukaemia virus type 1 p21X mRNA: constitutive expression in peripheral blood mononuclear cells of patients with adult T cell leukaemia

Although the p21X protein of human T cell leukaemia virus type 1 (HTLV-1) is generally thought to be expressed from a doubly spliced mRNA transcript (tax/rex mRNA) that encodes the p40tax, p27rex and p21X proteins, we have shown previously that a novel, alternatively spliced mRNA transcript (p21X mRNA) is responsible for p21X production in HTLV-1-infected cell lines. In the present study, we analysed expression of p21X mRNA and tax/rex mRNA in uncultured and cultured peripheral blood mononuclear cells (PBMCs) from eight patients with adult T cell leukaemia by using a quantitative polymerase chain reaction coupled to reverse transcription. The results demonstrated that the expression of p21X mRNA occurs constitutively in all uncultured and cultured PBMCs, whereas the expression of tax/rex mRNA is inducible in the cultured PBMCs, as described previously. In uncultured and cultured PBMCs from the one specimen in which p21X mRNA was highly expressed, the p21X protein was detectable by Western blotting. On the other hand, p27rex protein was detectable only after cultivation. These findings indicate that p21X mRNA is constitutively expressed in vivo and is responsible for production of p21X protein.

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.

Rapid, sensitive, specific, and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers.

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01–0.025 µM. This increases sensitivity and specificity enough to detect from 1 to 105 copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.

Identification of novel singly spliced pX mRNA transcripts common to all human T-cell leukemia virus type 1-related retroviruses

Human T-cell leukemia virus type 1 (HTLV-1) and its related viruses, that is, human T-cell leukemia virus type 2 (HTLV-2), simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV), encode a doubly spliced pX mRNA transcript in addition to the singly splicedenv and unsplicedgag-pol mRNAs common to the prototypic simple retroviruses, such as murine and avian leukemia viruses. In HTLV-1-infected cells, we recently identified the novel singly spliced pX mRNA responsible for expressing the smallerrex gene product, p21X. In the present study we demonstrate that the novel singly spliced pX mRNA is also expressed in cells infected with HTLV-2, STLV, and BLV, respectively. This finding indicates that all members of the HTLV-1-related virus group have the common ability to express the singly spliced pX mRNA. This novel mRNA in the HTLV-1-related virus group may be analogous to the two-exonnef specific mRNA in human immunodeficiency virus type 1.