A METHOD FOR EXTRACTION OF HIGH MOLECULAR WEIGHT DNA FROM THE MACROALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1,2

We developed a method for extraction of DNA from the red alga Porphyra yezoensis Ueda. The method consists of three preparation steps that include CsCl-gradient ultracentrifugation, cetyl trimethyl ammonium bromide treatment, and a final RNase step. The amount of DNA extracted from 1.5 g of starting material averaged 17.7 μg. The resulting DNA had a high molecular weight, was 25-166 kb in length, was digested with five common restriction enzymes, and showed no nuclease activity. It was of sufficient quality for construction of genomic libraries.     

Determination of concentrations of flecainide in human serum by high-performance liquid chromatography on a fluorocarbon-bonded silica gel column

An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C₁₈ column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 μl; column temperature, 40°C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength=300 nm, emission wavelength=370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide–acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10–2000 ng/ml). The regression equation was y=0.08+0.0078x (r=0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94–100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.     

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines

Immunogenic cell death induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4⁺ and CD8⁺ T cells producing interferon-γ. In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum, but whose functional relevance is elusive. Here, we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines. Chemotherapy reduced the number of tumor-infiltrating B cells, and failed to promote humoral responses against immunodominant tumor antigens. Although anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B-cell-depleting anti-CD20 antibody), immunoglobulins (Igs) or Ig receptors (Fc receptor) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells post chemotherapy (calreticulin, CRT) did not significantly increase in patients treated with anthracyclines, and that anti-CRT antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in, thus far, that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.     

Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-A(y)/Ta mice

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-β1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.     

Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor

Two assay methods for recombinant human tumor necrosis factor (rH-TNF) were developed, one a biological L-cell assay and the other an enzyme-linked immunosorbent assay. The accuracy and reproducibility of each and the correlation between the two were studied. As a result of this investigation, the two assay methods were found appropriate for standardization of rH-TNF. A freeze-dried reference was prepared, and examination of its potency and stability showed it to be suitable for use as a reference standard for rH-TNF assays.     

Stand structure and regeneration of <Emphasis Type="Italic">Populus euphratica</Emphasis> forest in the lower reaches of the Heihe River,NW China

Populus euphratica Oliv. is a main tree species that forms natural riparian forests in arid and semi-arid areas from Morocco to the Ordos Plateau. This study is designed to clarify the forest structure and dynamics of P. euphratica and to elucidate the ecological mechanisms sustaining riparian forests under unreliable environmental conditions. This study was conducted in a P. euphratica forest of the Ejina Oasis in Inner Mongolia, China, which is a hyperarid area. According to their tree size distribution, P. euphratica forests can be grouped into juvenile, mature, and overmatured stages. Almost all large P. euphratica showed dieback. The regeneration density on the forest floor shows a relation with the degree of height decrease due to dieback damage, as evaluated using the ratio of actual height to the maximum height estimated from the D–H relation. Therefore, after the mature stage, individual trees continue to grow while controlling their canopy size to adjust to changing environmental conditions in the overmatured stage. Our results suggest that P. euphratica growing under large fluctuations in groundwater levels exhibit a sophisticated regeneration system with canopy degradation.     

Fourier-transform infrared studies on conformation changes in bd-type ubiquinol oxidase from Escherichia coli upon photoreduction of the redox metal centers.

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli that does not belong to the heme-copper terminal oxidase superfamily. To explore unique protein structural changes associated with the reduction of the redox metal centers, we carried out Fourier-transform infrared and visible spectroscopic studies on cytochrome bd. For infrared measurements of a partially dehydrated thin sample solution, the air-oxidized enzyme was fully reduced by the intermolecular electron transfer of photo-excited riboflavin in the absence and presence of KCN, and redox difference spectra were calculated. Upon reduction, the bound cyanide was released from the heme b595-heme d binuclear center but remained in a protein pocket as a deprotonated form. Reduction of heme b558, heme b595, and heme d resulted in large changes in amide-I and protonated carboxylic CO-stretching vibrations and also a small change in the cysteine SH-stretching vibration. The location of the redox metal centers and the effects of cyanide suggest that these protein structural changes occur at the heme-binding pockets near the protein surface. Systematic site-directed mutagenesis and time-resolved FTIR studies on cytochrome bd will facilitate an understanding of the unique molecular mechanisms for dioxygen reduction and delivery of chemical protons to the active center at the atomic level.     

Nature of Cu(II)-poly(glutamic acid) complex in aqueous solution

Complex formation of poly(glutumic acid) with cupric ions in aqueous solution was investigated by three different methods: optical spectroscopy, optical rotatory dispersion, and electron spin resonance. Formation of a characteristic complex was found to occur in a pH region suitable for the helix-coil transition. An analysis of the ESH spectrum of the complex is given, and the results of calculation of bonding parameters suggest that the bond between copper and nitrogen atoms had an appreciably covalent character. The change in the secondary structure of the polymer as a result of complex formation is discussed.