Future target molecules for influenza treatment.

Induction of apoptosis and pro-inflammatory cytokine gene expression in influenza virus-infected cells activates production of toxic superoxide by macrophages. Pyrrolidine dithiocarbamate and nordihydroguaiaretic acid inhibit influenza virus proliferation and scavenge superoxide. These results suggest that they can be potential candidates for a drug of choice for influenza chemotherapy.

Secretion of bioactive interleukin-6 and tumor necrosis factor-alpha proteins from primary cultured human fetal membrane chorion cells infected with influenza virus.

Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection.

Improvement of separation method of fragmented DNA from an apoptotic cell DNA sample for the quantitation using agarose gel electrophoresis.

In order to quantify fragmented DNA extracted from apoptotic cells, we devised a separation method which condenses fragmented DNA into a small band, separating it from larger-size DNA with agarose gel electrophoresis. Calf thymus DNA and standard fragmented DNA were loaded onto 1.0% gel for 0.5, 1.0, 1.5 and 2.0 cm length, and onto 0.7, 1.0, 1.5 and 2.0% of gels for 1 cm length. DNA was then extracted from gel slices with the UltraClean 15 DNA Purification Kit, and estimated by measuring fluorescence intensity using Hoechst No.33258 dye. DNA recovery from the gel showed constant values regardless of the amount of loaded DNA up to 1 microg/assay, and a plot of loaded DNA amounts vs. the DNA amount yielded resulted in a strait line in any gel concentration used. Our results show the best conditions to estimate DNA fragmentation rates in apoptotic cells in which fragmented DNA was separated from thymus DNA by loading on 1.0% gel for 1.0 cm length. We used our method to estimate fragmentation rates in DNA fractions extracted from apoptotic human cervical fibroblast, amnion epithelial and chorion laeve trophoblast cells by stimulation with actinomycin D. The results show that DNA fragmentation rates in these cells were consistent with the electrophoretic patterns of the DNA samples shown by their photographs.

[Apoptotic cell death induced by actinomycin D arisen at different levels and cell cycle stages according to human cultured cell species].

Nine kinds of human cultured cells, including fetus cells (smooth chorion trophoblast cells, amnion epithelial cells and HE-21), adult non-carcinoma cells (HCF), and carcinoma cells (KATO-III, COLO 201, Lu-134-AH, SK-OV-3 and SKG-3a) were stimulated with Actinomycin (Act.) D for 24 h. Apoptosis induction was investigated by agarose gel electrophoresis for DNA fragmentation analysis and by flow cytometric analysis of stained cells using in situ terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling TUNEL) staining techniques for the quantification of apoptosis, and simultaneously using propidium iodide for the gain of some information about cell cycle. By agarose gel electrophoresis, DNA fragmentation of these cells except amnion epithelial and SKG-3a cells was detected, depending on concentration of Act. D. Using flow cytometric analysis, these cells were separated into four groups according to the information about cell cycle. Group 1 included amnion epithelial and SKG-3a cells, which were TUNEL negative. In group 2, all cell populations at G0/G1 and G2/M phases of HCC, KATO-III and SK-OV-3 were TUNEL staining positive. A portion of each G0/G1 or G2/M phase cell of Lu-134-AH and COLO 201 in group 3 was TUNEL stain positive. In group 4, G2/M phase cells of smooth chorion trophoblast cells and HE-21 were mostly stained and a small population of G0/G1 phase cells were also TUNEL stain positive. These results show that the stages of the cell cycle at which apoptosis was arisen by Act. D stimulation were significantly different depending on the cells types.

Biosynthesis of Vicia graminea lectin- and Vicia unijuga lectin-binding glycoproteins in human tumor and nontumor cells and an estimation of its epitope structure.

We investigated biosynthesis of Vicia graminea lectin (VGA)- and Vicia unijuga lectin (VUA)-binding (Vgu) glycoproteins, which are human malignant tumor-associated antigens, in cultured human tumor and non-tumor cells by pulse-labeling experiments with [35S]-methionine, followed by immunoprecipitation using immobilized VUA, SDS-PAGE and autofluorography. It was shown that Vgu glycoproteins synthesized by tumor cells were 15-30 times greater than those of non-tumor cells. It was also shown that about 40-70% of Vgu glycoproteins synthesized by non-tumor cells were secreted from the cells while more than 80% of the antigen synthesized by tumor cells was not secreted, and that Vgu glycoproteins consisted of multiple molecular species with the same epitope. To estimate the epitope structure of Vgu glycoproteins, in preliminary experiments we prepared sialoglycoproteins and/or sialoglycopeptides from purified human glycophorin A. Human glycophorins A(M) and A(N) (GPs-A(M) and A(N)) were treated with Clostridium perfringens neuraminidase to remove all sialic acid residues linked to carbohydrate chains, with Newcastle disease virus (NDV) to remove alpha2-3 linked sialic acid residues, and by Edman's degradation to eliminate N-terminal amino acid of GP-As. Partial or complete desialylation reactions resulted in disappearance of the reactivity of GP-A(M) and GP-A(N) with corresponding antisera and in appearance of reactivities with VUA and VGA. Elimination of N-terminal amino acid of GP-As also resulted in appearance of reactivities with VUA. These results show that sialoglycoproteins with similar serological properties of Vgu glycoprotein could be prepared from GP-As, and suggest that the epitope structure of Vgu glycoprotein may be related to the MN blood type-epitope structure and its sialic acid residues at N-terminal moiety of GP-As.

Presence of Vicia graminea lectin- or Vicia unijuga lectin-binding (Vgu) glycoproteins, Vgu glycoproteins with Thomsen-Friedenreich (T) activity and T-active glycoproteins in human meconium.

Perchloric acid-soluble fraction prepared from a mixture of meconiums of 22 newborn babies was subjected to a systematic affinity chromatography using Vicia unijuga lectin-Cellulofine column and Arachis hypogaea lectin-Agarose column and separated into four fractions, Vgu glycoprotein, Vgu glycoprotein with T activity, T-active glycoprotein and another glycoprotein fractions. HPLC analysis showed that the first fraction contained three glycoproteins with molecular weight (Mw) of about 19,400 kDa, 5500 kDa and 1900 kDa, the second fraction consisted of two glycoproteins with Mw of about 460 kDa and 150 kDa and the third fraction composed of several glycoproteins including the main glycoprotein with Mw of about 11,700 kDa.

Maxillofacial fractures in children.

A clinico-statistical and long-term follow-up study was performed on 81 pediatric fractures seen during the 14 years between 1977 and 1990. Of all maxillofacial fractures, the incidence of pediatric fractures was 14.7%. The ratio of boys to girls was 2.1:1, and the highest incidence involved boys over 13 years of age. Fractures of the upper alveolar bone and mandible were common. Conservative therapy, such as maxillomandibular fixation using orthodontic brackets was usually performed and was found to be successful. The long-term follow-up study revealed that 5 out of 21 patients with alveolar fractures complained of malocclusion and it is suggested that a longer duration of intramaxillary fixation and long-term follow-up might be needed for alveolar fractures in children.

[Serological property of perchloric acid-soluble glycoprotein fractions of human meconium].

Perchloric acid-soluble fractions (PASFs) were separated, in yields of 87.0-151.9 mg per 1.0 g of wet meconium sample, from six samples of human meconium, and identified as glycoproteins having 41.2-74.1% carbohydrate by chemical composition analysis. Six PASFs reacted with anti-A, -B, -Lea and -Leb sera, but did not react with anti-M and -N sera. These PASFs alos reacted with many lectins such as DBA, UEA-I, Vicia graminea lectin (VGA), Vicia unijuga lectin (VUA), Con A, WGA, RCA-I, PHA-E, SBA, SJA, and influenza A and B viruses. The serological results show that six PASFs contained VGA or VUA-binding (Vgu) glycoproteins as new oncofetal substances, but did not contain M and N blood group substances.

[Clinical study of maxillofacial fractures sustained during sports and games].

We performed a clinico-statistical study of the maxillofacial fracture due to sports during the period of 15 years between 1977 and 1991. Eighty-nine patients were seen with injuries resulting from 21 different sports. The incidence of the fracture was most common in rugby, followed by ski, baseball and soccer. Males were shown to be more prone to maxillofacial fracture than females (5.4:1) and the highest incidence of injuries involved the 20-29 age group followed by the 10-19 age group. The major parts of the fracture were the mandible and the alveolar process. Conservative therapy such as maxillomandibular fixation was usually performed and the clinical course was good. It was suggested that the prevention of the sports-related fracture and the time of returning to sports after the fracture will require further study.