Gen-Ichiro Soma

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa β-galactoside-binding lectin

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.

Production and purification of a recombinant human 14 kDa β-galactoside-binding lectin

The cDNA for a 14 kDa human β‐galactoside‐binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single‐step chromatography on an asialofetuin‐agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N‐terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N‐terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three‐dimensional structure as the placental lectin.

Absence of a γ-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta

Abstract 1. 1. Complete nucleotide sequence of one of the salmon proopiomelanocortin mRNAs (POMC mRNAs) was determined. 2. 2. The region corresponding to γ-melanocyte-stimulating hormone (γ-MSH) was lacking in salmon POMC mRNA, although overall organization of the multi-hormone structure was exactly the same as that of mammalian POMC mRNAs. 3. 3. The possible evolutional history of POMC mRNA in mammalian species may be revealed from the finding of this characteristic that salmon POMC mRNA lacks the region corresponding to γ-MSH.

Purification of rabbit tumor necrosis factor.

Rabbit tumor necrosis factor (TNF) was purified and shown by SDS‐PAGE to be a single protein of 18 kDa. TNF in 355 ml rabbit serum was precipitated with ammonium sulfate, and purified by repeated DEAE‐Sephadex and Sephacryl S‐200 chromatographies, and the final fractionation on Blue‐Sepharose 6B. By this procedure its yield was 22% and its specific activity was 2.4 × 107 U/mg protein. The sequence of the N‐terminal 20 amino acids was determined.

A LARGE NUMBER OF TANDEM REPEATS IN THE POLYMORPHIC EPITHELIAL MUCIN GENE IS ASSOCIATED WITH SEVERE ACNE

Polymorphic epithelial mucin (PEM) or MUC1 is a glycoprotein secreted from various epithelial gland tissues. In skin, PEM is detected in sweat glands and sebaceous glands by the DF3 monoclonal antibody. The gene of PEM includes an allele exhibiting length polymorphism due to a variable number of tandem repeats (VNTR); this is expressed co‐dominantly, which may influence the microenvironment of the skin. The allelic size variation of the PEM gene was investigated in Japanese acne patients, atopic dermatitis patients, and healthy controls. The frequency of longer length alleles was significantly higher in severe acne patients.

Primary structure of two mRNAs encoding putative salmon α-subunits of pituitary glycoprotein hormone

1. By random sequence analyses, we isolated from the cDNA library of salmon pituitary glands two clones, the deduced amino acid sequences corresponding to the C-terminal region of which are almost the same as those of the alpha subunits of mammalian glycoprotein hormones. 2. Comparison of the nucleotide sequences and deduced amino acid sequences from these two clones with those of mammalian species revealed that the two newly-isolated cDNAs corresponded to mRNAs encoding the putative salmon pre-alpha subunit of glycoprotein hormones. 3. Homology in the nucleotide sequences of these two clones suggested that corresponding mRNAs may be encoded by separate genes which probably evolved from a common ancestral gene.

Improvement of cytotoxicity of tumor necorosis factor (TNF) by increase in basicity of its N-terminal region

Two new recombinant TNFs (named rTNF-Scw1 and -Scw2) with higher basicity than conventional recombinant human TNF-alpha (rTNF-alpha) in the N-terminal region were constructed. Their sequences were constructed based on those of partially purified cytotoxic factors from the culture supernatant of acute monocytic leukemia cells THP-1, which unlike rTNF-alpha are cytotoxic to T24 bladder carcinoma cells in vitro. These new rTNF-Ss showed a broader cytotoxicity to tumor cells than rTNF-alpha. This increase in the basicity of the N-terminal region over that of conventional TNF significantly increased the cytotoxicity on tumor cells in vivo as well as in vitro.

Hemodynamic evaluation of recombinant human tumor necrosis factor (TNF)-α, TNF-SAM2 and liposomal TNF-SAM2 in an anesthetized dog model

Summary We have evaluated the hemodynamic effects of systemically administered recombinant human tumor necrosis factor (TNF)-α, TNF-SAM2 and liposome-bound TNF-SAM2 in an anesthetized mongrel dog model. A dose of 10 μg TNF protein/kg of each formulation was injected in a peripheral vein and mean systemic arterial pressure (SAP), heart rate (HR) and cardiac output (CO) were measured. TNF-α induced a marked drop in SAP in all three dogs (mean decrease = 59.3 ± 5.2 mm Hg; to 61.5% of baseline; p = 0.008); whereas TNF-SAM2 caused a smaller and transient drop in SAP in four dogs (mean decrease = 25.5 ± 10.1 mm Hg; to 81.2% of baseline; p = 0.086). In three dogs administered liposome-bound TNF-SAM2, which retains antitumor activity in vivo, a net slight hypertensive phase and sustained elevated CO occurred, followed by a return to an essentially normotensive state (101.0% of baseline SAP). This model demonstrates that the principal acute systemic toxicity of TNF, i.e., hypotension, can be markedly attenuated by liposomal formulation of a second-generation TNF.

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

SummaryThe human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D3 [1α, 25(OH)2D3] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10−8M 1α,25(OH)2D3 alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)2D3 was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)2D3 might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5-1000 micrograms/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 micrograms/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 micrograms/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.