Characteristics of Alkyltrimethoxysilane-Treated Fumed Silicas and Rheological Behavior of Fumed Silica Suspensions in an Epoxy Resin.

Fumed silica was surface treated by hexyl (C6), octyl (C8), decyl (C10), hexadecyl (C16), and octadecyl(C18)trimethoxysilanes, and the products were evaluated from various aspects. The amounts of alkyl group introduced on silica surface were all within the range from 22 to 25 mmol/100 g. Such fumed silicas were suspended in an epoxy resin, bisphenol type A, and their rheological properties were measured. The rheological behavior of the silica suspensions was investigated as to steady-state viscosity and dynamic moduli in order to examine the effect of alkyl chain length on interactions of hydrophobic groups on the silica surface. The stronger shear thinning behavior was observed in the surface-modified fumed silica with longer alkyl groups. However, the suspensions of the nontreated fumed silica and the fumed silicas treated by C6 silane showed Newtonian behavior even at the silica concentration of 7 wt%. The dynamic moduli G' of the silica suspensions increased with increasing alkyl chain length. The interaction of hydrophobic groups on the silica surface is mainly attributed to the rheological behavior of fumed silica suspensions in the epoxy resin. Copyright 2001 Academic Press.

Amino-terminal fragment of urokinase-type plasminogen activator inhibits HIV-1 replication.

CD8+ T lymphocytes have been shown to produce unidentified soluble factors active in suppressing HIV-1 replication. In this study, we purified an HIV-1 suppressing activity from the culture supernatant of an immortalized CD8+ T cell clone, derived from an HIV-1 infected long-term nonprogressor, and identified this activity as the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA). ATF is catalytically inactive, but suppresses the release of viral particles from the HIV-1 infected cell lines via binding to its receptor CD87. In contrast, cell proliferation and the secretion of an HIV-1 LTR driven reporter gene product were not affected by ATF. These findings suggest that ATF may inhibit the assembly and budding of HIV-1, which provides a novel therapeutic strategy for AIDS.

A newly developed assay for melatonin using cells expressing human mel-1a receptor.

We have developed a radioreceptor binding assay (RRA) method for melatonin using membranes from Chinese hamster ovary cells that can stably express human mel-1a receptors. We measured melatonin levels in plasma samples collected every 4h for 24h using the RRA and radioimmunoassay (RIA) methods, simultaneously. There was a statistically significant correlation between the melatonin levels measured by the two methods, this newly developed method providing a sensitive bioassay. As it is possible to circumvent the cross-reactivity usually occurring in the RIA method, this method may be an important tool for detecting bioactive substances relative to the mel-1a receptor.

Interleukin-9 receptor alpha chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s).

A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-1) activity of CD8+ T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8+ T cells and the interleukin-9 receptor alpha chain (IL-9R-alpha) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-1 activity of CD8+ T cell culture supernatants was assessed by measuring the level of HIV-1 replication of a CD4+ T cell line transfected with an infectious HIV-1 DNA clone. IL-9R-alpha mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-1 activity (more than 80% suppression of HIV-1 replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-1 activity (less than 80% suppression of HIV-1 replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-1 replication at a concentration of 1 microgram/ml. These data suggest that the IL-9R-alpha mRNA formation in CD8+ T cells may correlate with and play some role in the anti-HIV-1 activity of CD8+ T cells from HIV-1-infected individuals.

Immunopotentiating activity of the water-soluble lignin rich fraction prepared from LEM--the extract of the solid culture medium of Lentinus edodes mycelia.

The water-soluble lignin in LEM (the extract of the solid culture medium of Lentinus edodes mycelia) has been known to have antiviral and immunopotentiating activities in vivo and in vitro. The water-soluble lignin rich fraction (JLS-18) was prepared from LEM using ultrafiltration and hydrophobic column chromatography. JLS-18 showed about 70 times higher antiviral activity than LEM in vitro. JLS-18 activated the cytotoxicity of NK cells and macrophages, and activated T cells in vitro. JLS-18 also induced interleukin 6 (IL-6) secretion from human leukocytes infected with Sendai virus in vitro. These data showed that JLS-18, the water-soluble rich fraction of LEM, had antiviral and immunopotentiating activities.

Biologically inactive growth hormone caused by an amino acid substitution.

Short stature caused by biologically inactive growth hormone (GH) is characterized by lack of GH action despite high immunoassayable GH levels in serum and marked catch-up growth to exogenous GH administration. We found a heterozygous single-base substitution (A-->G) in exon 4 of the GH-1 gene of a girl with short stature, clinically suspected to indicate the presence of bioinactive GH and resulting in the substitution of glycine for aspartic acid at codon 112. We confirmed the presence of mutant GH in the serum using isoelectric focusing analysis. The locus of mutation D112G was found within site 2 of the GH molecule in binding with GH receptor (GHR)/GH binding protein (GHBP). The expressed recombinant mutant GH tended to form a 1:1 instead of the 1:2 GH-GHBP complex normally produced by wild-type GH. The formation of a 1:2 GH-GHBP complex is compatible with the dimerization of GHRs by GH, a crucial step in GH signal transduction. Mutant GH was less potent than wild-type GH not only in phosphorylation of tyrosine residues in GHR, janus kinase 2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in IM-9 cells, but also in metabolic responses of BaF/GM cells, a stable clone transfected with cDNA of the chimera of the extracellular domain of human GHR, the transmembrane and the cytoplasmic domain of the human thrombopoietin receptor. These results indicate that the D112G mutation in the GH-1 gene causes production of bioinactive GH, which prevents dimerization of GHR and is therefore responsible for the patient's short stature.

Identification of glycosylated subtypes of interferon-alpha produced by human leukocytes.

We found that interferon-alpha produced by human leukocytes contained four subtypes that were glycosylated by N-linkages according to the results of lectin blot analysis. These glycosylated subtypes were type H or type omega and their sugar moieties had no relation to their biological activities.

Existence and unique N-terminal sequence of alpha II (omega) interferon in natural leukocyte interferon preparation.

Human alpha interferon produced in Sendai virus-stimulated leukocytes was purified by immunosorbent affinity chromatography and subjected to subtype analysis. All subtypes in leukocyte culture supernatant were confirmed in purified preparation. One of these subtypes was reactive in Western blotting with antibody specific to alpha II (omega). N-terminal amino acid sequence analysis of this particular subtype showed addition of two amino acids to the N-terminus of alpha II predicted through cDNA studies. The apparent cleavage site for leader sequence of alpha II was different from the site common to all other alpha subtypes. This is the first report on the isolation of natural alpha II and its unique N-terminal amino acid sequence.

Studies on subtype composition in natural leukocyte interferon preparations.

Antisera raised against partially purified human leukocyte interferon in goat and horse were exhaustively adsorbed and purified. Affinity chromatography using these antibodies gave 10,000-fold purification in one step without losing interferon activity. Then enzyme immunoassay was established with these antibodies and utilized for subtype analysis. Crude and affinity purified leukocyte interferons showed identical profiles of antiviral activity and immunoreactivity when fractionated by means of reversed phase high performance liquid chromatography. These results suggested the possibility of providing mixture of subtypes of leukocyte interferon occurring in natural host response, with comparable purity of recombinant ones.

Monoclonal antibodies specific for high molecular weight form of human epidermal growth factor.

Hybridomas that secrete monoclonal antibodies specific for the high molecular weight (HMW) form of human epidermal growth factor (hEGF) were established by fusing spleen cells obtained from mice immunized with purified urinary HMW-hEGF with myeloma P3 x 63Ag8.653. The resulting monoclonal antibodies were characterized basically into two groups. One group recognized both EGF and HMW-hEGF, while the other recognized HMW-hEGF specifically on radio immunoprecipitation. Surprisingly, the majority of the isolates was positive by western blotting. Utilizing these monoclonal antibodies for affinity chromatography, we purified HMW-hEGF successfully from urine. These antibodies may be an extraordinarily powerful tool for histological study related to both forms of EGF.