Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

Isolation of two forms of decay-accelerating factor (DAF) from human urine.

Decay-accelerating factor (DAF) was purified from human pooled urine by conventional techniques. The urine DAF was separated into two peaks, pool I and pool II, by gel chromatography. DAF-U1 was isolated from pool I by hydrophobic chromatography, and DAF-U2 from pool II by anti-DAF IgG column. The specific activities of DAF-U1 and DAF-U2 to decay membrane-phase C5 convertase were about 3% and 70% of membrane form DAF, respectively. However, both urine DAFs revealed a similar activity to each other and slightly higher activity than that of membrane form DAF in decay-accelerating fluid-phase C3 convertase of the alternative pathway.     

Monoclonal antibody against platelet thrombospondin decreases erythrocyte aggregation rate.

The effect of thrombospondin, a major glycoprotein in the platelet alpha-granule, on the erythrocyte aggregation rate was investigated. Venous blood was sampled from 8 healthy male volunteers and anticogulated with 1.1 mg/ml EDTA(K2). The erythrocyte aggregation rate of each blood sample was measured with a whole-blood erythrocyte aggregometer before and after incubation with murine monoclonal antibody against human platelet thrombospondin. After 15 min incubation, the erythrocyte aggregation rate exhibited a significant decrease to 0.055 +/- 0.022/s, representing 71.9 +/- 8.7% of the control value (0.075 +/- 0.028/s) (p less than 0.0005). The results obtained suggest that thrombospondin may participate in the control of erythrocyte aggregability in the circulating blood.     

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.     

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.     

Selective production of hybridoma cells: antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells

Methods for pre-selecting B lymphocytes were studied and investigated. First, biotinylated antigen was used for selecting B lymphocytes. These pre-selected B lymphocytes were then combined with biotinylated myeloma cells by adding streptavidin. The final formula of the selected B cell-myeloma cell was as follows: B cell-(antigen-biotin-strept-avidin-biotin)-myeloma cell. Then, this B cell-myeloma cell conjugate was fused by the pulsed electric field (PEF) method, which fused only those conjugated cells. The fusion efficiency obtained by this method was 3-15-times higher than that obtained by the non-specific poly(ethylene glycol) (PEG) fusion method. Second, avidin-antigen conjugate was used to select B lymphocytes. For this purpose, bifunctional cross-linkers such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and m-maleimidobenzoyl N-hydroxysuccinimide (MBS) were chosen. Each reagent contains two heterofunctional groups which can make covalent bond with both Lys and Cys residues. Typical avidin-antigen conjugate is expressed as avidin-SPDP (or MBS)-antigen. Thus, final B cell-myeloma cell conjugate was B cell-antigen-SPDP (or MBS)-avidin-biotin-myeloma cell. The yield of this procedure was of the order of 10(-2). Here, we suggest that the pre-selection of B lymphocytes by biotinylated antigen or avidin-antigen conjugate is a new method of obtaining selected hybridoma cells which produce specific monoclonal antibodies against the antigen used for selecting B lymphocytes.     

Reproductive lifespan and reproductive performance in SPF C3H mice: the onset of reproductive life and production efficiency (author's transl)]

To improve the production system, the onset and the termination of reproductive life of C3Hf/HeMsNrs mice mated immediately after weaning and reared for 400 days of life, were studied. From weaning females mated with a full grown male (group A), the first litter was obtained at a mean age of 47 days, suggesting the first copulation at 26 days of age. The age of males at the first copulation was estimated to be at 44 days of age from the age giving the first litters in weanling males mated with weanling (group B) and full grown (group C) females. The sex ratio of litters delivered by young dams tended to be excess in males. The reproductive performance of dams in later life was not affected by the parturition in earlier age. The production efficiency with weaned youngs per pair during the first 200 days after mating was the highest in group A. It was found from these results that the C3H females attained their sexual maturity at 5 to 6 days after weaning, being available for breeding without any deletion in reproductive performance.     

Fluorimetric assay of sialic acids.

A fluorimetric assay has been developed for sialic acids in which sialic acids react with pyridoxamine to give fluorescent compounds in the presence of zinc ion and pyridine. This assay method is specific for unbound sialic acids and is a simple and sensitive procedure compared with the thiobarbituric acid assay of sialic acids.     

Crystal and molecular structure of 6,2'-anhydro-1-beta-D-arabinofuranosylctyosine.

The crystal and molecular structure of the title compound has been determined by X-ray diffraction method. The compound crystallizes in monoclinic system with the space group P2₁ and Z=2; the unit cell dimensions are a=10.491, b=7.255, c=6.858 A and β=103.55°. The structure was refined to an R-index of 0.051. The glycosyl torsion angle X_CN is 111.4° (syn-anti) and the arabinose ring forms an exo-conformation, in which C(4′) is displaced by 0.61 A out of the plane of remaining four atoms. The orientation of the C(5′)O(5′) bond is the gauche-gauche as similar as that found frequently in many nucleosides.