Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte, Microsporum canis.

A homolog of the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa was isolated from the zoophilic dermatophyte, Microsporum canis. This gene, dnr1, encodes a polypeptide of 761 amino acid residues containing a single zinc-finger DNA-binding domain, which is almost identical in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional equivalence of dnr1 to areA was demonstrated by complementation of an areA loss-of-function mutant of A. nidulans with dnr1 cDNA. To further characterize this gene, dnr1 was disrupted by gene replacement based on homologous recombination. Of 100 transformants analyzed, two showed the results expected for replacement of dnr1. The growth properties of the two dnr1(-) mutant strains on various nitrogen sources were examined. Unlike the A. nidulansareA(-) mutant, these dnr1(-) mutants showed significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These mutant strains were also able to utilize various amino acids for growth. In comparison with wild-type M. canis, the two dnr1(-) mutants showed reduced growth on medium containing keratin as the sole nitrogen source. This is the first report describing successful production of targeted gene-disrupted mutants by homologous recombination and their phenotypic analysis in dermatophytes.     

N-Glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase proteins of human parainfluenza virus type 4A and 4B

cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant proteins, and found that the antigenicities of the mutant hPIV-4B HN proteins were more similar to the hPIV-4A HN protein than to the non-mutant hPIV-4B HN protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes. Received: 21 January 2000     

Characterization and expression of cDNA encoding coproporphyrinogen oxidase from a patient with hereditary coproporphyria

Hereditary coproporphyria (HCP) is an acute hepatic porphyriawith autosomal dominant inheritance, but with a variable degreeof clinical expression. Molecular cloning, sequencing and expressionof the defective gene for coproporphyrinogen oxidase (CPO) ina patient with HCP were carried out. Enzyme assays revealedthat CPO activity in EBV-transformed lymphoblastoid cells fromthe proband and one of her sisters was     

Astroglial responses against Abeta initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey.

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.     

ALPHA FETOPROTEIN and GARGINOEMBRYONIC ANTIGEN PRODUCING HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma (HCC) showing marked elevation of serum alpha fetoprotein (AFP) (maximum; 70942.0 ng/ml at the end stage) and serum carcinoembryonic antigen (CEA)(maximum; 7368.4 ng/ml at the end stage) was surgically resected. In the resected liver, there were two different tumor nodules which were adjacent to each other but clearly separated by a thin connective tissue. One of the nodules was a well differentiated and the other was poorly differentiated HCC. Immunoperoxidase study revealed that both CEA and AFP were localized in the tumor cells of the poorly differentiated HCC. This is the first report which clearly proved CEA synthesis in the cells of HCC. Serial staining showed that there was simultaneous synthesis of CEA and AFP in some of the tumor cells. ACTA PATHOL. JPN. 35: 969–974, 1985.     

An autopsy case of acquired immune deficiency syndrome (AIDS) with preceding aplastic anemia

A case of acquired immunodeficiency syndrome (AIDS) with preceding aplastic anemia is reported. The patient was a 36 year old female who had been diagnosed as having aplastic anemia 10 years before and thereafter had received multiple transfusions. Human immunodeficiency virus (HIV)-seropositivity was revealed 10 months prior to her death, but no particular clinical signs indicating HIV infection, pre-AIDS or onset of AIDS were recognized before serological diagnosis, although the slow progression of leukopenia was noted along with thrombocytopenia. Her general condition deteriorated during the last 10 months accompanied by an acute decrease In the CD4/CD8 ratio. Autopsy revealed full-blown AIDS: systemic aspergillosis, progressive multifocal leukoencephalopathy, Epstein-Barr virus-related B cell lymphoma arising in the diaphragm and severe lymphocyte depletion in the lymph nodes and spleen. Markedly hypo-plastic bone marrow was considered to be primarily attributable to the aplastic anemia but the affection of AIDS was not excluded. The possible transmission route of HIV and the effect of the preceding aplastic anemia on the infection and clinical course of AIDS are discussed.     

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats

The copper and Iron status in the liver of non-tumor bearing Long-Evans Cinnamon (LEC) rats (average age 17 months) was investigated. A direct quantitation of loosely-bound copper and iron was also investigated by using a chelating agent, nitrilotriacetic acid (NTA-chelatable free copper and iron). Besides the total copper and iron contents, the level of NTA-chelatable free copper was also higher in LEC rats than In LEA rats (P<0.05). But for the free iron level there was no signiflcant difference between the two rat groups (P>0.05). The formation of thiobarbituric acid-reactive substances was higher In LEC rats than In LEA rats (P<0.01). The 4–hydroxy-2–nonenal (HNE)-modified proteins were also clearly demonstrated in LEC rat liver. The copper and iron which produced the most important effect In the process of oxidative damage in LEC rats could not be distinguished. Even though free copper, which could induce free radical injuries, was increased in LEC rats, neither tumor-induction nor preneo-plastic lesions in the experimental LEC rats were observed. Therefore it is speculated that the elevation of a free iron is another important factor. Copper and iron, both important translation metals In the body, may participate In the Induction of DNA damage and oncogenesls     

Immunoelectron microscopic localization of carcinoembryonic antigen in gastric adenocarcinoma cell lines

The distribution of carcinoembryonic antigen (CEA) in human gastric adenocarcinoma cell lines (HPE-GAC-3 cells and HPE-GAC-2 cells) was determined immunohistochemically by indirect peroxidase-labeled antibody method at the light and electron microscopic levels. In GAC-3 cells that proliferated as non-adherent single cells, CEA was located in the perinuclear spaces, the endoplasmic reticulum, Golgi apparatus, vesicles, multivesicular body (MVB) and entire plasma membrane. Membrane CEA was shown to be internalized into MVB in GAC-3 cells. In GAC-2 cells that form an acinus, CEA was predominantly present along the microvilli of the lumina) surface and in glycocalyceal bodies, the vesicles which bud from the microvilli into the lumen. These results suggest that in poorly differentiated cancer cells CEA is transported over the entire cell surface, retained on the membrane and accumulated Into the cell by way of the MVB, but in well differentiated cancer cells the newly synthesized CEA is rapidly and predominantly transported to the luminal surface and rapidly released from the membrane into the lumen by way of the glycocalyceal body.     

Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain

Background: We previously reported that the homozygous mutation of Otx2 gene, a mouse cognate of the Drosophila head gap gene orthodenticle , causes failure in the development of the rostral head anterior to rhombomere 3, which may correspond to earlier Otx2 expression in cells destined for the anterior mesoendoderm. At the same time, the Otx2 heterozygous mutation displayed a phenotype characterized as otocephaly, probably related to expression in the anterior neuroectoderm at the subsequent pharyngula stage. Defects were characteristic in the most anterior and posterior regions of Otx2 expression where Otx1 , another mouse cognate of orthodenticle , is not or weakly expressed. They were not found in the region where Otx1 is expressed.
Results: In the present work, Otx1 null mutant mice were generated by gene targeting in embryonic stem cells. No defects were apparent in the regionalization of the early embryonic rostral brain. The newborn brain defects were subtle and most likely related to later Otx1 -unique expression. Otx1 and Otx2 double heterozygous mutant brains, however, exhibited marked defects throughout the fore- and midbrains, where defects were not apparent with a single mutation alone.
Conclusions: Otx1 and Otx2 play synergistic roles in the development of the forebrain and midbrain where both genes are expressed.