The injection of plasmid DNA in mouse muscle results in lifelong persistence of DNA, gene expression, and humoral response

The duration of the immune response against any vaccine is critical. The present study was performed to determine the stability of injected plasmid deoxyribonucleic acid (DNA), the duration of gene expression in mouse muscle, as well as the duration of the immune response generated in mice after injection of plasmid pSO2C1 harboring the cry11Bb gene of Bacillus thuringiensis serovar. medellin. The localization and the persistence of the inoculated gene were determined by in situ hybridization and polymerase chain reaction (PCR). The results demonstrated that plasmid DNA can persist in mouse muscle for up to 2 yr. Moreover, immunohistochemical analysis showed that Cry11Bb protein was expressed for the lifetime of the mice at a low but significant level. Finally, production of Cry11Bb-specific antibodies in mice injected with pSO2C1 was high and durable as significant antibody titers were observed up to 119 wk after injection of the plasmid. This persistent immune response is likely owing to the existence of a protein and/or DNA depot in the organism, which serves to maintain the immune response, acting as a secondary or booster immunization.     

The C-terminal domain of Escherichia coli MutY is involved in DNA binding and glycosylase activities

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxo-guanine (8-oxoG). The C-terminal domain of MutY is required for 8-oxoG recognition and is critical for mutation avoidance of oxidative damage. To determine which residues of this domain are involved in 8-oxoG recognition, we constructed four MutY mutants based on similarities to MutT, which hydrolyzes specifically 8-oxo-dGTP to 8-oxo-dGMP. F294A-MutY has a slightly reduced binding affinity to A/G mismatch but has a severe defect in A/8-oxoG binding at 20°C. The catalytic activity of F294A-MutY is much weaker than that of the wild-type MutY. The DNA binding activity of R249A-MutY is comparable to that of the wild-type enzyme but the catalytic activity is reduced with both A/G and A/8-oxoG mismatches. The biochemical activities of F261A-MutY are nearly similar to those of the wild-type enzyme. The solubility of P262A-MutY was improved as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. The binding of GB1-P262A-MutY with both A/G and A/8-oxoG mismatches are slightly weaker than those of the wild-type protein. The catalytic activity of GB1-P262A-MutY is weaker than that of the wild-type enzyme at lower enzyme concentrations. Importantly, all four mutants can complement mutY mutants in vivo when expressed at high levels; however, F294A, R249A and P262A, but not F261A, are partially defective in vivo when they are expressed at low levels. These results strongly support that the C-terminal domain of MutY is involved not only in 8-oxoG recognition, but also affects the binding and catalytic activities toward A/G mismatches.     

Microampullary organs of a freshwater eel-tailed catfish,Plotosus (tandanus) tandanus

Whole body studies of Plotosus tandanus revealed that ampullary pores occur over the entire body of the fish, but are in higher concentrations in the head region. These pores give rise to a short canal (50-60 microm) produced by columnar epithelial cells bound together by tight junctions and desmosomes. At the junction of the canal and the ampulla, cuboidal epithelial cells make up the wall. The ampulla consists of layers of collagen fibers that surround flattened epithelial cells in the lateral regions and give rise to supportive cells that encase a small number of receptor cells (10-15). The ampullary wall comprises several types of cells that are adjoined via tight junctions and desmosomes between cell types. The ovoid receptor cells possess microvilli along the luminar apical area. Beneath this area, the cells are rich in mitochondria and rough endoplasmic reticulum. An unmyelinated neuron adjoins with each receptor cell opposite multiple presynaptic bodies. This form of microampulla has not been previously described within the Family Plotosidae.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

Induction, purification, and characterization of two extracellular alpha-L-arabinofuranosidases from Fusarium oxysporum

In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two alpha-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed of three equal subunits and are neutral proteins with pIs of 6.0 and 7.3 for ABF1 and ABF2, respectively. With p-nitrophenyl alpha-L-arabinofuranoside (pNPA) as the substrate, ABF1 and ABF2 exhibited Km values of 0.39 and 0.28 mmol.L(-1), respectively, and Vmax values of 1.6 and 4.6 micromol.min(-1).(mg of protein)(-1), respectively, and displayed optimal activity at pH 6.0 and 50-60 degrees C. ABFs released arabinose only from sugar beet arabinan and not from wheat soluble and insoluble arabinoxylans. The enzymes were not active on substrates containing ferulic acid ester linked to C-5 and C-2 linkages of pNPA showing that phenolic substituents of pNPA sterically hindered the action of ABFs.     

Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia

Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans. Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition.     

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.     

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS-PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.     

Solution properties of an alpha-(1-->3)-D-glucan from Lentinus edodes and its sulfated derivatives

A water-insoluble alpha-(1-->3)-D-glucan (A) from Lentinus edodes was fractionated into 13 fractions in dimethyl sulfoxide containing 0.25 M lithium chloride (0.25 M LiCl-Me(2)SO). Five fractions were treated with sulfur trioxide-pyridine complex at 25 degrees C to synthesize water-soluble sulfated derivatives (S-A). The weight-average molecular weights, M(w), and intrinsic viscosities [eta], of the samples A and S-A were determined by multi-angler laser light scattering (MALLS), and viscosity. The M(w) dependence of [eta] and of the radius of gyration (z)(1/2), was found to be represented approximately by [eta]=4.9 x 10(-2) M(w)(0.67) (cm(3) g(-1)), and (z)(1/2)=4.8 x 10(-2) M(w)(0.54) (nm) for the alpha-glucan in 0.25 M LiCl-Me(2)SO in the M(w) range from 7.24 x 10(4) to 4.21 x 10(5), and by [eta]=6.8 x 10(-4) M(w) 1.06 (cm(3) g(-1)), and (z)(1/2)=9.4 x 10(-4) M(w)(0.92) (nm) for the sulfated alpha-glucan in aqueous 0.5 M NaCl in the M(w) range from 5.92 x 10(4) to 1.42 x 10(5) at 25 degrees C. The results indicate that the alpha-(1-->3)-D-glucan exists as a flexible chain in 0.25 M LiCl-Me(2)SO, and its sulfated derivative in 0.5 M NaCl aqueous has stiffer chains than the original. (13)C NMR indicated that intramolecular hydrogen bonding occurred in the sulfated alpha-glucan, causing the observed chain stiffness.