Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.     

A case of small polypoid esophageal carcinoma with multidirectional differentiation, including neuroendocrine, squamous, ciliated glandular, and sarcomatous components

A small composite esophageal carcinoma measuring 1.5 x 1.4 x 1.0 cm is described. The tumor had a polypoid elevation with a superficial extension. Histologic examination revealed invasion of the submucosal layer and multidirectional differentiation, including neuroendocrine, squamous, ciliated glandular, and sarcomatous components. The neuroendocrine component was strongly positive for chromogranin and formed the bulk of the polypoid tumor. The squamous cell carcinoma exhibited a superficial extension. The adenocarcinoma was located in a small region of the tumor and contained ciliated glandular cells. The spindle cell sarcomatous component, which was positive for alpha-smooth muscle actin and negative for cytokeratin, exhibited no specific mesenchymal differentiation. Each component was found in 60%, 10%, 5%, and 25% of the tumor, respectively. Cases of small composite esophageal carcinoma containing various carcinomatous and sarcomatous components are extremely rare.     

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.     

Atomic force microscopy to study direct neurite-mast cell (RBL) communication in vitro

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL-2H3) cells. Atomic force microscopy (AFM) showed how neurites attached to a pseudopodium or a cell body of an RBL cell. After stimulation of SCG neurites with bradykinin or scorpion venom, RBL cells attached to neurites spread and flattened, and several discharged granules (0. 5-1.0 microm in diameter) were found on the surface of the RBL cells. A neurokinin (NK)-1 receptor (i.e. substance P receptor) antagonist prevented the RBL degranulation. The results showed that activation of the SCG neurites with bradykinin or scorpion venom was able to elicit degranulation in RBL cells which were attached to neurites.     

Humanization of a mouse neutralizing monoclonal antibody against tumor necrosis factor-alpha (TNF-alpha)

An anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-alpha. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-alpha antibody is expected to be less immunogenic and thus more suitable for possible clinical use.     

Apoptosis signaling pathways in lung diseases

Evidence that apoptosis plays an important role in the pathophysiology of lung diseases has been accumulated. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. Death receptor ligation triggers recruitment of the precursor form of caspase-8 to a death-inducing complex, through the adaptor protein FADD, which leads to caspase-8 activation. The other pathway triggered by stimuli such as drugs, radiation, infectious agents and reactive oxygen species is initiated in mitochondria. After cytochrome c is released into the cytosol from the mitochondria, it binds to Apaf1 and ATP, which then activate caspase-9. Recently, endoplasmic reticulum has also been shown to be the organelle to execute apoptosis. Further understanding of molecular mechanisms of apoptosis and its regulation by novel drugs may lead to the development of effective strategies against lung diseases. We overview the signaling pathways of apoptosis and discuss the involvement of apoptosis in the pathophysiology of various lung diseases.     

Conversion of a gene-specific repressor to a regional silencer

In Saccharomyces cerevisiae, gene silencing at the HMR and HML loci is normally dependent on Sir2p, Sir3p, and Sir4p, which are structural components of silenced chromatin. Sir2p is a NAD+-dependent histone deacetylase required for silencing. Silencing can be restored in cells lacking Sir proteins by a dominant mutation in SUM1, which normally acts as a mitotic repressor of meiotic genes. This study found that mutant Sum1-1p, but not wild-type Sum1p, associated directly with HM loci. The origin recognition complex (ORC) was required for Sum1-1p-mediated silencing, and mutations in ORC genes reduced association of Sum1-1p with the HM loci. Sum1-1p-mediated silencing also depended on HST1, a paralog of SIR2. Both Sum1-1p and wild-type Sum1p interacted with Hst1p in coimmunoprecipitation experiments. Therefore, the SUM1-1 mutation did not change the affinity of Sum1p for Hst1p, but rather relocalized Sum1p to the HM loci. Sum1-1-Hst1p action led to hypoacetylation of the nucleosomes at HM loci. Thus, Sum1-1p and Hst1p could substitute for Sir proteins to achieve silencing through formation of a compositionally distinct type of heterochromatin.     

Combination chemotherapy with cyclophosphamide, adriamycin, cisplatin, nimustine(ACNU), and methotrexate (EACAM) in advanced adenocarcinoma of the lung

Twenty-two patients with advanced adenocarcinoma of the lung were treated with the combination chemotherapy "EACAM" consisting of cyclophosphamide (333mg/m2 X 1), adriamycin (27mg/m2 X 1), cisplatin (25mg X 5), nimustine (33mg/m2 X 1), and methotrexate (27mg/m2 X 3). This regimen was repeated once every 4 or 5 weeks. One complete response (CR) and 8 partial responses (PR) were obtained in 21 evaluable patients and the response rate was 42.9%. It has not been possible to calculate the median survival time for all of the evaluable cases, since 13 of them are still alive up to the present time. The side effects observes were as follows: nausea and vomiting (81.8%), alopecia (81.8%), stomatitis (22.7%), leukocytopenia less than 2,000/mm3 (45.5%), and thrombocytopenia less than 5 X 10(4)/mm3 (18.2%). Apart from strong myelosuppression, no severe infection or bleeding tendency was noticed. A mild elevation of serum createnine was observed in one patient, and no patients developed renal insufficiency. The combination chemotherapy "EACAM" is therefore considered to be a very effective and tolerable treatment for adenocarcinoma of the lung.