How should xanthine oxidase-generated superoxide yields be measured?

The rate of formation of superoxide measured by its reduction of tetranitromethane (TNM) and by its reduction of ferric cytochrome c (Fe(III) cc) are in excellent agreement when the superoxide is generated from a simple chemical precursor. In contrast, the rate of formation of superoxide generated in the reaction of xanthine oxidase with acetaldehyde is much higher (up to a factor of 6) when measured with TNM and compared with Fe(III) cc. It is shown that Fe(III) cc measures superoxide that has diffused from the enzyme, and that TNM probably scavenges all the dioxygen that is reduced by one electron by the enzyme. The TNM traps enzyme-bound superoxide in competition with the second-electron transfer and proton transfer, which normally yield hydrogen peroxide. The proton transfer is probably rate determining, k(p) 相似文献   

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics. Journal of Industrial Microbiology & Biotechnology (2000) 25, 333–341. Received 27 April 2000/ Accepted in revised form 25 November 2000     

Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus)

To date, it is not known whether the seasonal occurrence of sexual behavior and mating in free-ranging Hanuman langurs at Ramnagar, Nepal, is correlated with seasonal changes in female ovarian function, and, if so, which factor(s) triggers the onset of the reproductive period. Using noninvasive fecal hormone analysis in combination with behavioral observations, this study was carried out to: 1) investigate and characterize seasonal patterns of ovarian cyclicity and timing of conception in wild langur females living in a highly seasonal habitat; and 2) examine the relationship between seasonal patterns of ovarian cyclicity, behavioral estrus, and female physical condition. Behavioral data and fecal samples were collected during a total period of 14 months from nine females living in a multi-male group. Physical condition of the females was assessed monthly by visual inspection, using a seven-fold scale. Ovulatory cycles and timing of conceptions were identified by the measurement of immunoreactive pregnanediol glucuronide (iPdG) in extracted feces. Hormone profiles in individual females revealed a clearly seasonal distribution in the occurrence of ovulatory cycles, which were restricted to the period from July to October. The distribution of female estrus behavior showed a similar seasonal pattern, and in total 88.2% of all estrus periods observed in the focal females were accompanied by ovulation. Onset of ovarian cycles as well as mating activities were strongly correlated with the onset of the rainy season. Females conceived, on average, in their second ovulatory cycle (pregnancy length: 211.6 +/- 3.4 days), with timing of conception being confined to the months when animals showed an improved physical condition. Collectively the present data clearly suggest that in seasonally-breeding langurs at Ramnagar, ecological conditions (rainfall, food availability, and quality) influence the onset of ovulations and timing of conceptions.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Prophylactic and therapeutic effects of Mytilus edulis fatty acids on adjuvant-induced arthritis in male Wistar rats

Lipid-rich fractions from the flesh tissue of Mytilus edulis were obtained by solvent extraction and chromatographic separation, and tested for anti-inflammatory (AI) activity in vitro and in vivo. Inhibition of leukotriene production by isolated human neutrophils in response to calcium ionophore stimulation in the presence of exogenous arachidonic acid substrate was demonstrated for the hydrolysed triglyceride fraction of the crude lipid extract. This fraction was subsequently tested for in vivo AI activity using the mycobacterial adjuvant-induced polyarthritis rat model. The hydrolysed triglyceride fraction showed significant AI activity when dosed therapeutically (10 mg/kg BW/day, p.o., for 6 days from the onset of arthritis), decreasing body weight loss by 55% and hind paw swelling by 65% compared to the arthritic control. The (non-hydrolysed) crude lipid extract was effective when dosed prophylactically (30 mg/kg BW/day, p.o., for 16 days starting on day ?2 of arthritigen inoculation). Structural analysis by GC and GC–MS revealed in the extracts an abundance of EPA (20:5n-3) and DHA (22:6n-3) (37% of total fatty acids), along with a small quantity of a rare anti-inflammatory n-3 analogue of arachidonic acid, namely 7, 11, 14, 17-eicosatetraenoic acid (20:4n-3).     

Long-term data on basic reproductive parameters and evaluation of endocrine,morphological, and behavioral measures for monitoring reproductive status in a group of semifree-ranging Tonkean macaques (Macaca tonkeana)

Cycle and gestation lengths, menstruation patterns, female genital swelling characteristics, and male-female consortship durations are reported in a semifree-ranging group of Tonkean macaques (Macaca tonkeana) studied over a 12 year period. In addition, profiles of urinary estrone conjugates (E1C) and immunoreactive pregnanediol glucuronide (PdG) throughout four complete menstrual cycles in two females and three full-term pregnancies are presented. Based on intermenstrual intervals, a mean cycle length of 37–41 days (n = 55 cycles in 10 females) was found. Gestation length averaged 173 days (n = 27 pregnancies in eight females). Measurement of PdG immunoreactivity in urine revealed a cyclic pattern with a 5–15-fold increase between follicular and luteal phase concentrations, suggesting that PdG is a reliable indicator of ovarian cyclicity and luteal function. In contrast to PdG, E1C excretion showed no clear pattern throughout the cycle; however, highest values of E1C were usually found shortly before the onset of the luteal phase PdG rise at the presumed time of ovulation. Levels of both hormones were elevated during the first half of gestation and showed a marked increase throughout the second half, with maximum E1C concentrations being up to 100-fold higher than nonpregnant levels. Consortships by the male and occurrence of female genital swelling were long lasting (on average 5–10 days and 13 days, respectively) and were restricted to the follicular phase of the cycle. The day of maximal swelling and day of detumescence as well as the end of male consortship were closely associated with the periovulatory period. Swellings and consortships were longer following lactational ammenorhea than for subsequent cycles. The evolutionary significance of the cyclical changes undergone by females upon their relations with males is discussed. © 1996 Wiley-Liss, Inc.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.