Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

VARIATION IN THE ULTRASTRUCTURE OF THE BIFLAGELLATE MOTILE CELLS OF SIX UNICELLULAR GENERA OF THE CHLAMYDOMONADALES AND CHLOROCOCCALES (CHLOROPHYCEAE), WITH EMPHASIS ON THE FLAGELLAR APPARATUS

The ultrastructural features of the biflagellate motile cells of six different species of the Chlorophyceae, namely Dunaliella lateralis (Polyblepharidaceae, Chlamydomonadales), Chlorococcum hypnosporum, Spongiochloris spongiosa, Protosiphon botryoides (Chlorococcaceae, Chlorococcales), Tetracystis aeria and Pseudotetracystis terrestris (Tetracystidaceae, Chlorococcales), were examined with an emphasis on the flagellar apparatus (FA). They have different vegetative characteristics, such as, being motile or nonmotile, variations in chloroplast morphology, possession of one or more nuclei, and reproductive features such as formation of tetrahedral tetrads, and naked or walled zoospores. Ultrastructural differences amongst reproductive cells of the six species include variations in cell surface structure, basal body to basal body angle, beamlike extensions of the distal fiber, extensive connections of the proximal sheath between basal bodies, two-membered rootlets, striated microtubule-associated components, two-membered rootlet-nucleus and/or mitochondria connections, X-membered rootlets, connections of rootlets and basal bodies, rhizoplasts and accessory basal bodies. All six species possess pyrenoids penetrated by thylakoid membranes, and the FA typical of the Chlorophyceae (sensu Mattox and Stewart, 1984). These six species should be divided into two groups. The first includes D. lateralis, C. hypnosporum, and T. aeria, in which accessory basal bodies are present, the basal body to basal body angle is relatively fixed, and a cell wall or surface coat is present. The second group includes Ps. terrestris, S. spongiosa, and Pr. botryoides, in which accessory basal bodies are absent, the basal body to basal body angle is variable and the zoospores are naked.     

Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C

We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.     

Molecular cloning of cDNA for proteasomes (multicatalytic proteinase complexes) from rat liver: primary structure of the largest component (C2)

Proteasomes (multicatalytic proteinase complexes) from rat liver are composed of at least 13 nonidentical components [Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, M., Sato, M., Tanaka, N., Katsube, Y., Kameyama, K., & Takagi, T. (1988) J. Mol. Biol. 203, 985-996]. The nucleotide sequence of one major component (C2) of the proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a mixture of synthetic deoxyribonucleotides as a probe. The sequence was composed of 1174 nucleotides including a coding region for the entire protein and noncoding regions of both the 5'- and 3'-sides. The polypeptide deduced from the open reading frame consisted of 263 amino acid residues, and its molecular weight was calculated to be 29,516. The partial amino acid sequences of several fragments (approximately 45% of the total residues), which were obtained by cleavage of C2 with lysyl endopeptidase and cyanogen bromide, were determined by automated Edman degradation and found to be in complete accordance with those deduced from the cDNA sequence. The amino acid composition of C2, determined by chemical analysis, was also consistent with that deduced from the cDNA sequence, indicating that the cloned cDNA actually encoded component C2. Computer analysis revealed little structural similarity of C2 to other proteins reported so far. Northern blot hybridization analyses showed that the mRNA encoding this novel protein C2 was expressed in all the rat tissues examined and in a variety of eukaryotic organisms such as amphibia, birds, and mammals with slight species-specific differences in size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxanthine guanine phosphoribosyltransferase deficiency: nucleotide substitution causing Lesch-Nyhan syndrome identified for the first time among Japanese

Summary A previously undescribed nucleotide substitution at codon 51 (CGA to TGA) has been identified using the polymerase chain reaction technique in hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA; this is the first molecular evidence for a point mutation in a Japanese patient with Lesch-Nyhan syndrome. The present mutation is the 19th nucleotide substitution identified as a germ-line mutation at this locus and the second mutation generating a stop codon. The position of the nucelotide substitution is exactly the same as a previously described mutation HPRT_Toronto, indicating for the first time that nucleotide substitutions at the same position in the sequence of HPRT can generate different mutant alleles, one causing a partial deficiency and the other a complete deficiency. Although the type of nucleotide substitution is different between the two cases, a single base position has twice become the target of a mutation. However, the calculation of the probability of finding substitution mutations at the same base position in the coding region of hprt indicates that there is no evidence for the presence of a hot spot for substitution mutations in the human hprt germ line.