Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver

Neural cell adhesion molecule (N-CAM) is distributed in most nerve cells and some non-neural tissues. The present immunohistochemical study has revealed, for the first time, the expression of N-CAM in perisinusoidal stellate cells of the human liver. Liver specimens were stained with monoclonal antibody against human Leu19 (N-CAM) by a streptoavidin-biotin-peroxidase-complex method. Light- and electron-microscopic analyses have shown that N-CAM-positive nerve fibers are distributed in the periportal and intermediate zones of the liver lobule. Perisinusoidal stellate cells in these zones are also positive for N-CAM. N-CAM is expressed on the surface of the cell, including cytoplasmic projections. Close contact of N-CAM-positive nerve endings with N-CAM-positive stellate cells has been observed. On the other hand, stellate cells in the centrilobular zone exhibit weak or no reaction for N-CAM. Perivascular smooth muscle cells and fibroblasts in the portal area and myofibroblasts around the central veins are negative for N-CAM. The present results indicate that the perisinusoidal stellate cells in the periportal and intermediate zones of the liver lobule characteristically express N-CAM, unlike other related mesenchymal cells, and suggest that the intralobular heterogeneity of N-CAM expression by stellate cells is related to the different maturational stages of these cells.     

Functional evaluation of the adenohypophysis with metoclopramide in hyperprolactinemic-amenorrheic women in relation to radiological findings on the sella turcica

The responses of the adenohypophyseal hormones to metoclopramide (MCP) were evaluated in hyperprolactinemic women with various radiological findings on the sella turcica. Serum PRL concentrations significantly increased after MCP administration in normal women, hyperprolactinemic patients with normal sella and patients with microadenoma, but not in macroadenoma patients with and without suprasellar expansion (SSE). The PRL response to MCP administration was significantly lower in hyperprolactinemic patients than in normal women. Serum TSH concentrations significantly increased after MCP administration in each group of subjects. The TSH response to MCP was significantly higher in patients with normal sella and patients with microadenoma than in normal women. However, the responses of PRL and TSH to MCP were not significantly different between patients with normal sella and patients with microadenoma. Therefore, they were not considered useful in distinguishing tumorous from nontumorous hyperprolactinemia. Serum LH concentrations significantly increased after MCP administration in patients with normal sella, patients with microadenoma and macroadenoma patients without SSE, but not in normal women or macroadenoma patients with SSE. The LH response to MCP was significantly higher in patients with microadenoma than in patients with normal sella. Serum FSH concentrations significantly increased after MCP administration only in patients with microadenoma. The different responses of the adenohypophyseal hormones to MCP in hyperprolactinemic women with various radiological findings on the sella turcica may be explained by the difference in the hypothalamic dopamine activity and in the impairment of the hypothalamic-pituitary system due to pituitary tumor.     

Alteration of a DNA-dependent ATPase activity in xeroderma pigmentosum complementation group C cells.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks by fast protein liquid chromatography Mono Q column chromatography. Similar elution profiles were observed with the extracts from human cells normal in repair and xeroderma pigmentosum cells belonging to complementation groups A through G except for group C. An alteration in elution of one of the five ATPases, designated DNA-dependent ATPase Q1, was observed with a cell line of complementation group C. This alteration was observed with all tested cell lines that belonged to group C. ATPase Q1 in HeLa cell extracts exhibited about 2-fold higher activity with ultraviolet light-irradiated DNA as compared to that with non-irradiated DNA, whereas little difference in the effects of two DNAs was observed with the ATPase activities in the extract from group C cells.     

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Accumulation of vanadium during embryogenesis in the vanadium-rich ascidian,Ascidia gemmata

It is a remarkable and previously unrecognized fact that ascidians, which are known to contain high levels of vanadium in their blood cells, begin to accumulate vanadium during embryogenesis. This study revealed that the accumulation starts quite dramatically 2 wk after fertilization, and 2 mo later, the amount of vanadium in larvae is 600,000 times higher than that in the unfertilized egg. These results were obtained by neutron activation analysis, a highly sensitive method for determining levels of vanadium, in theAscidia gemmata, the ascidian that contains the highest known levels of vanadium and accumulates vanadium at 150 mM in its blood cells, a concentration that corresponds to 4,000,000 times the concentration in sea-water.     

Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein. The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function. The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function. Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells. This molecular-shift appeared depending on an activation of p34cdc2 kinase.