Dual function for U2AF(35) in AG-dependent pre-mRNA splicing.

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.

Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis.

A megakaryoblastic cell line, termed T-33, was established from the peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis. T-33 cells have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for over 2 years. Giemsa-banded karyotypes were female hyperdiploid with a modal chromosomal number of 51, all cells including Philadelphia chromosome. The cells showed strong positivity for periodic acid-Schiff and alpha-naphthyl acetate esterase, and weak for alpha-naphthyl butyrate esterase, but were negative for myeloperoxidase. Flow cytometric analysis of cell surface markers showed the existence of HLA-DR, MY-7, MY-9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes. Most of the cells fixed with acetone were positive for Factor VIII, platelet glycoprotein IIb-IIIa, IIIa (Yukb), and Ib, but negative for glycophorin A and hemoglobin. Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% after the treatment with 12-O-tetradecanoylphorbol-13-acetate. The cells contained small dense granules negative for platelet peroxidase, their number increasing threefold after 12-O-tetradecanoylphorbol-13-acetate treatment. Such treated cells frequently showed a complex of the demarcation membrane in the cytoplasm. T-33 responded thrombin to exhibit calcium influx. This cell line may be useful for the study of the early stage of megakaryocytic differentiation in human megakaryopoiesis.

Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. CLA has been reported to reduce body fat. To examine the mechanism(s) of CLA reduction of fat mass, female C57BL/6J mice were fed standard semipurified diets (10% fat of total energy) with or without CLA (1% wt/wt). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) and DNA fragmentation analysis revealed that fat-mass decrease by CLA was mainly due to apoptosis. Tumor necrosis factor (TNF)-alpha and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. Because it is known that TNF-alpha induces apoptosis of adipocytes and upregulates UCP2 mRNA, a marked increase of TNF-alpha mRNA with an increase of UCP2 in adipocytes caused CLA-induced apoptosis. However, with a decrease of fat mass, CLA supplementation resulted in a state resembling lipoatrophic diabetes: ablation of brown adipose tissue, a marked reduction of white adipose tissue, marked hepatomegaly, and marked insulin resistance. CLA supplementation decreased blood leptin levels, but continuous leptin infusion reversed hyperinsulinemia, indicating that leptin depletion contributes to the development of insulin resistance. These results demonstrate that intake of CLA reduces adipose tissue by apoptosis and results in lipodystrophy, but hyperinsulinemia by CLA can be normalized by leptin administration.

SF2/ASF binds to a splicing enhancer in the third HIV-1 tat exon and stimulates U2AF binding independently of the RS domain.

Splicing of a single HIV-1 primary transcript into more than 30 different mRNAs is regulated by a combination of suboptimal splice sites, cis-acting RNA splicing enhancers and silencers, and trans-acting factors. We have studied the splicing of the second tat intron (SD4 to SA7) and find that activation of splicing by SF2/ASF is mediated by a degenerate exon splicing enhancer (ESE3), consisting of at least three functionally independent sub-elements. One of these sub-elements appears to have both enhancing and silencing properties, depending on the context. SF2/ASF stimulates U2AF65 binding to the suboptimal tat polypyrimidine tract in an ESE3-dependent manner, whereas the exon splicing silencer (ESS3) that is located downstream of the ESE3 inhibits this step. Truncated SF2/ASF protein without the RS domain binds specifically to the ESE3 and retains almost full capacity to stimulate U2AF65 binding and activate splicing. This suggests that SF2/ASF can stimulate the recruitment of U2AF65 by an RS domain-independent mechanism.

Replacement of m-calpain by mu-calpain during maturation of megakaryocytes and possible involvement in platelet formation.

Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca(2+)-requiring form (m-calpain) and the low-Ca(2+)-requiring form (mu-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-mu-calpain, and staining with anti-mu-calpain was prominent only in megakaryocytes. To confirm the result, megakaryoblastic cell line (T-33) cells were subjected to immunoblot analysis. However no immunoreactivity to mu-calpain was seen in T-33 cells. Bone marrow from a patient with idiopathic thrombocytopenic purpura showed immature megakaryocytes (stage II) strongly stained by anti-m-calpain antibody while mature cells (stage III) were strongly stained by anti-mu-calpain antibody. These results suggest that mu-calpain plays a crucial role in mature megakaryocytes, possibly in platelet production.

Histopathologic studies on myeloproliferative sarcoma virus (MPSV) induced leukemias and hemangiosarcoma in Jar-2 rats.

It is well known that MPSV induces myeloproliferative syndrome (MPS) in mice. Intravenous one shot inoculation of myeloproliferative sarcoma virus (MPSV) with Friend murine leukemia virus (F-MuLV) as a helper in newborn Jar-2 rats (on the second neonatal day) yielded hematopoietic malignancies in all the treated rats (25/25 rats) after 2 weeks' latency. MPS appeared from the 14th day in 14 rats. In the midst of the myeloproliferative field of the spleen and bone marrow, myeloblastic or myeloblastic-erythroblastic foci were observed. From 19th day, acute myeloblastic leukemia occurred in 3 rats and erythroleukemia in 8 rats. MPSV induced first MPS which remained as such or later developed into acute leukemia. Myelofibrosis as seen in mice was not observed. In addition, hemangiosarcoma of the brain, spinal cord and spleen appeared in 15 rats from the 24th day, and were often multiple. MPSV can yield the tumor only in newborn rats, and target cells of MPSV are not only hematopoietic cells but also endothelial cells of the brain, spinal cord and occasionally spleen.

In vivo fluoride profiles at different sites of buccal and lingual enamel surfaces obtained by enamel biopsy of human maxillary first permanent molars in young adults.

Twenty male volunteers, average age 24 years, participated in this study. Specimens were obtained by enamel biopsy using 5 microliters of 0.5 M HClO4 for 30 s. Using a regression curve, comparisons of fluoride concentrations were made at different depths. The fluoride concentrations (mean +/- SE) at a depth of 5 microns were highest in the distobuccal (1698 +/- 136), high in the mesiobuccal (1343 +/- 122), low in the distolingual (1119 +/- 107), and lowest in the mesiolingual sites (819 +/- 78). Of the interior enamels (> or = 10 microns in depth), the distobuccal site (1330 +/- 88 parts/10(6) F at 10 microns) had a higher-concentration than all other sites. The fluoride profiles were steepest to shallowest in the order: distobuccal, mesiobuccal, distolingual and mesiolingual. There were no correlations between the enamel fluoride concentrations and the fluoride concentration in parotid saliva. It was concluded that in vivo fluoride profiles of maxillary first molars reflect the wear of the tooth surface with age and the condition of dental plaque deposition, and, to some extent, the site-specific distribution of saliva between buccal and lingual surfaces.

Fulminant septicemic syndrome of Bacillus cereus in a leukemic patient.

We report a rapidly fatal Bacillus cereus septicemia in a leukemic patient receiving remission-induction therapy. Symptoms resembling food poisoning and fever preceded coma accompanied by neurologic abnormalities. Autopsy revealed necrotizing leptomeningitis with subarachnoid hemorrhage and coagulation necrosis of the liver with bacterial infiltration. These clinicopathologic findings were closely similar to those of reported cases. Because of a rapidly fatal clinical course, suspicion of this syndrome early in the course is important to determine an appropriate treatment. Therefore, we propose that this type of septicemia should be termed as fulminant septicemic syndrome of Bacillus cereus.

Cyclosporine G and D in experimental autoimmune uveoretinitis in the rat.

Two analogs of cyclosporine A (CsA), cyclosporine G (CsG) and cyclosporine D (CsD), were compared to CsA with regards to their effects on experimental autoimmune uveoretinitis (EAU) as well as their adverse effects on renal functions. When administered daily on days 0-14 after immunization with S-antigen, CsA was the most effective of all in inhibiting EAU followed by CsG: 20-30 mg/kg/day of CsG appeared to have the same effect as 5 mg/kg/day of CsA. The effect of CsD was the least. When administered from day 7 after immunization, CsA and CsG were also effective in inhibiting the development of EAU. As for the adverse side effects, CsA was the most nephrotoxic: the toxic changes were morphologically found with doses of CsA at 10 mg/kg/day or higher. CsG and CsD were not nephrotoxic even at 30 mg/kg/day. The effects of CsG on immune responses were very similar to those of CsA. Both agents exhibited selective inhibition on the cell-mediated immune responses to S-antigen, while having no effect on antibody production.

Development of hybrid type artificial bone marrow using sintered hydroxyapatite.

In vivo inducement of hybrid-type artificial bone marrow with hemopoietic inductive microenvironment (HIM) in sintered hydroxyapatite (HA) chamber was carried out. This research is important to disclose the mechanisms of hemopoiesis and is useful for clinical application. In the evolution of vertebrates, cartilage of the inner skeleton changed into bone, having biomechanical properties to form bone marrow cavities. The hemopoietic nests immigrated into the cavities from the spleen. We should be able to induce hemopoietic nests in a hydroxyapatite chamber in place of bone, if we can find optimal structural conditions. Therefore, we tried to artificially induce a hematopoietic field in muscles using sintered porous tubular hydroxyapatite and new type hydroxyapatite plate made by high-pressure gas technique. As a result, not only in the pore sites of tubular hydroxyapatite artificial bone, but at the surface of the new type hydroxyapatite plate implanted in the dorsal muscles, marked differentiation of bone marrow cell clusters of the hematopoietic field could be observed.

[Mono-radiculopathy multiplex--multiple infarction of the cauda equina caused by intravascular lymphomatosis].

A 55-year-old man had felt numbness of the bilateral peroneal sides of legs for 6 months. Then hepatosplenomegaly, anemia, body weight loss and fever developed, and a diagnosis of malignant histiocytosis (MH) was made by revealing the presence of innumerable atypical histiocytes with hemophagocytosis in the bone marrow. Soon later, sensory disturbance of bilateral peroneal sides of legs (right side dominant) developed and aggravated with painful dysesthesia and weakness of the legs for the last 2 weeks before death. Electrophysiologically, sensory conduction velocity of the sural nerve was normal and somatosensory evoked potentials from tibial nerve were normal before P15 but were not evoked at all after the lumbar potential, suggesting lumbosacral radiculopathy. Autopsy showed multifocal ischemic lesions and secondary degeneration of the lumbosacral nerve roots associated with necrosis and fibrosis of the radicular vessels and intravascular infiltration of atypical mononuclear cells which were positive for B cell markers. The neurological manifestations and the distribution of ischemic lesions, which were similar to those of vasculopathic mononeuropathy multiplex, would deserve the name of "monoradiculopathy multiplex".

[A case of rhinosporidiosis].

Rhinosporidiosis is due to fungal infection by Rhinosporidium seeberi, which affects predominantly the mucous membrane of the nose and nasopharynx. This disease is characterized by the formation of papillomatous and polypoid lesions and is known to be endemic in India and Sri Lanka. The first case of rhinosporidiosis in Japan is reported in this paper. A 25-year-old male Indian, born in India, living in Japan for a year, visited the outpatient clinic complaining of nasal obstruction and swallowing pain. A friable, irregular, reddish polypoid mass was found in the right nasal cavity. Endoscopy as well as plain X-ray and CT examination showed that the mass originated from the right inferior turbinate and extended into the choana without any sign of bone destruction. The mass removed under general anesthesia showed the typical feature of rhinosporidiosis, that is, papillomatous hyperplasia of the mucosa with sporangia full of spores in different stages of development. One year after treatment, no relapse has been seen in this case.

Histopathological identification of hepatic intramitochondrial crystalloid inclusions (IMCI).

A 45-year-old female with systemic lupus erythematosus underwent liver biopsies twice during the period of cefazolin administration and a complication of cefazolin-induced toxic hepatitis was strongly suggested. Histologically, a small number of spindle-shaped, esoinophilic crystalloid bodies were faintly noticed in the cytoplasm of hepatocytes in hematoxylin and eosin-stained sections, while they showed strong positivity for strains inherent to mitochondria as well as specific to phospholipid and sharply showed a spindle or needle shaped configuration. Moreover, they greatly increased in number in these sections. They were distributed throughout the the parenchyma but were prominent in the degenerated hepatocytes with slight fatty metamorphosis. Electron microscopically, they proved to correspond to the intramitochondrial crystalloid inclusions (IMCI). Despite many ultrastructural descriptions of the IMCI, precise histopathological examination of the IMCI referring to the staining property on the deparaffinated sections has not yet been described since the first report of the IMCI by Napolitano (1958).