Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

Ultrafast protein determinations using microwave enhancement

We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine protein concentrations. Typical protein determinations involve incubation times ranging from 15–60 min. Microwave irradiation of specimens reduces this time requirement to 10–20 s without compromising accuracy or reliability. The remarkable speed with which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures that depend on the estimation of protein concentrations. An erratum to this article is available at .     

Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi . However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi , with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.     

Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.     

Poly(ADP-ribose) polymerase: Structural conservation among different classes of animals and its implications

Poly(ADP-ribose) polymerase cDNAs have been isolated from different classes of animals. Cloning of genes from lower eukaryotes has allowed us to investigate directly the biological functions of poly(ADP-ribosyl)ationin vivo. The conservation of specific regions among mammals, chicken,Xenopus laevis, andDrosophila melanogaster reveals the essential structural elements required for recognition of breaks in DNA and for catalytic activity. Cys, His and basic residues in the zinc-finger consensus region are conserved. The carboxyl terminal region corresponding to an NAD-binding domain is strongly conserved. The dinucleotide-binding consensus sequence and 1-A-2, Rossmann fold structure, and -sheet structures are completely conserved from mammals to insect. InDrosophila, a putative leucine-zipper motif has been identified, and other poly(ADP-ribose) polymerases also contain an -helical, amphipathic structure in the auto-modification domain. In this article, we review the recent structural analyses of the functional domains of poly(ADP-ribose) polymerase in phylogenetically divergent species, and discuss the implications of structural conservation for its biological functions.Abbreviations aa amino acid(s) - D. melanogaster Drosophila melanogaster - PARP poly(ADP-ribose) polymerase [EC 2.4.2.30] - PCR polymerase chain reaction - X. laevis Xenopus laevis     

Protein structure,electron transfer and evolution of prokaryotic photosynthetic reaction centers

Photosynthetic reaction centers from a variety of organisms have been isolated and characterized. The groups of prokaryotic photosynthetic organisms include the purple bacteria, the filamentous green bacteria, the green sulfur bacteria and the heliobacteria as anoxygenic representatives as well as the cyanobacteria and prochlorophytes as oxygenic representatives. This review focuses on structural and functional comparisons of the various groups of photosynthetic reaction centers and considers possible evolutionary scenarios to explain the diversity of existing photosynthetic organisms.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - Rb Rhodobacter - Rp Rhodopseudomonas     

PCR detection of MLOs in quick decline-affected pear trees in Italy

Polymerase chain reaction (PCR) amplification, using primers derived from the 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) analysis with Alu I restriction endonuclease was used to detect myc-oplasma-like organisms (MLOs) associated with pear decline. MLOs were consistently detected in pear trees that suddenly wilted and died within a few days during summer, as well as in pears of the same orchards with symptoms similar to the slow form of pear decline. In both cases the same RFLP pattern was obtained. Declining pear trees were 5 to 8-yr-old cvs Williams, Kaiser and Max Red Bartlett grafted on to Pyrus communis seedling rootstocks. All the orchards affected by quick decline had severe attacks of pear psyllid (Cacopsylla pyri) during the year this study was performed and during the previous year. The results showed the suitability of DNA amplification by the polymerase chain reaction for the detection of pear decline MLOs and established that MLOs can be detected in infected tissues of dead trees.     

精子介导鱼类基因转移和聚合酶链反应检测技术

金鱼精子与美洲大绵ｗｅｉ的抗冻蛋白基因一起保温３０分钟后,再与卵子受精,共获得１４５尾成鱼和若干胚胎。从胚胎和成鱼中提取ＤＮＡ经聚合酶链反应（ＰＣＲ法）扩增和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分子杂交表明,外源的抗冻蛋白基因进入了部分受体鱼的染色体组内。测定了４５尾一年龄实验鱼中,有１２尾显示出明确的杂交带,阳性率为２６％。     

Anaerobic acidogenesis of primary sludge: the role of solids retention time

This research investigates the effect of solids retention time (SRT) on the acid-phase anaerobic digestion of primary sludge. A series of experiments were conducted using two continuous-flow 3-L units with the following configuration: a completely mixed reactor (CMR) with clarifier and solids recycle and an upflow anaerobic sludge blanket (UASB) reactor. Results show that C(2) to C(5) volatile fatty acids (VFA) were the predominant compounds formed. At a constant hydraulic retention time (HRT) of 12 h, variation in SRT from 10 to 20 days resulted in a slight increase in VFA production in both systems, but at a shorter SRT (5 days) a drastic drop in acid production was observed. In addition, the percent distribution of VFA was to some extent affected by the change in SRT. On the other hand, organic matter degradation [measured by the chemical oxygen demand (COD) specific solubilization rate or the percent volatile suspended solids (VSS) reduction] appeared to be independent of SRT, at least in the range investigated. The percent soluble COD in the form of VFA, however, increased steadily with increasing SRT, approaching the 90% level at 20 days. The remaining soluble COD in the effluent from these systems may be mainly attributed to metabolic intermediates and unused soluble substrate. (c) 1994 John Wiley & Sons, Inc.     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.