Use of an automated sequencer to determine the sequence specificity of DNA damage

When searching on Scots pines, females of the aphid parasitoid Pauesia silvestris responded to differences in mortality risks, host distribution and host quality by changing foraging tactics. They foraged more successfully (i.e. they laid more eggs per unit time) on the pine aphid Cinara pini than on Cinara pineaTherefore, the former species was considered to be of higher quality. However, P. silvestris suffered from a high mortality (19.5%) from ant aggression when foraging for C. piniwhile mortality was zero on pines with C. pineaAll females that were killed were foraging on the bark, while females searching on needles were safe from ant attacks. When searching for C. pineaP. silvestris spent significantly more time on needles if the aphid colonies were ant-attended. On pines with C. piniin contrast, females spent more time on bark in ant-attended colonies. The high adult mortality risk on bark was counterbalanced by a significantly higher foraging success in ant-attended colonies.     

Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates

A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.     

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.     

Guidance method to use mixed coal in blending for coking

Based on the principle that the adaptation can be reflected by the overlap of reflectance distribution peaks,the effect of various types mixed coal for coking is analyzed.Based on the action of the vitrinite of different reflectance range and the adaptation,a new method for guidance blending coal is established.Through simulation,blending coal using the software of HD automatic microscope photometer,makes the synthetic blending coal reflectance distribution map to nothing notch wide single peak flat-shap...     

Artificial neural network method for predicting the specificity of GalNAc-transferase

The specificity of GalNAc-transferase is consistent with the existence of an extended site composed of nine subsites, denoted by R4, R3, R2, R1, R0, R1', R2', R3', and R4', where the acceptor at R0 is either Ser or Thr to which the reducing monosaccharide is anchored. To predict whether a peptide will react with the enzyme to form a Ser- or Thr-conjugated glycopeptide, a neural network method--Kohonen's self-organization model is proposed in this paper. Three hundred five oligopeptides are chosen for the training site, with another 30 oligopeptides for the test set. Because of its high correct prediction rate (26/30 = 86.7%) and stronger fault-tolerant ability, it is expected that the neural network method can be used as a technique for predicting O-glycosylation and designing effective inhibitors of GalNAc-transferase. It might also be useful for targeting drugs to specific sites in the body and for enzyme replacement therapy for the treatment of genetic disorders.     

Decomposition of the mixed rare earth concentrate by microwave-assisted method

A novel process was proposed to strengthen the decomposition of the mixed rare earth concentrate by utilizing the microwave radiation.Mineralogical information on the mechanisms by which microwave heating improved the leaching behavior of rare earth elements(REEs),and an interpretation of the interrelationship between mineralogy,decomposition process,and leaching process were provided in this study.The influences of the temperature,time of microwave heating and contents of NaO H(mass ratio of NaO H to mixed rare earth concentrate)on the decomposition of mixed rare earth concentrate were investigated.The results revealed that the temperature was the main factor affecting the decomposition process.The recovery of REEs by hydrochloric acid leaching reached 93.28% under the microwave heating conditions:140 oC,30 min and 35.35% NaO H.The BET specific surface area and SEM analysis indicated that the particles of mixed rare earth concentrate were non-hole,while the particles presented a porous structure after heating the concentrate by microwave radiation.For the microwave treated sample after water leaching,the BET specific surface area was 11.04 m~2/g,which was higher than the corresponding values(6.94 m~2/g)for the mixed rare earth concentrate.This result could be attributed to the phase changes of bastnaesite and monazite,and a number of cracks induced by thermal stress.The increase of BET specific surface area resulted in an increase of the recovery of REEs by promoting interaction within the system of acid leaching.     

用微波快速测量球团矿预配水量的新方法

对国内外几种测量水分的方法进行了评述,提出了用微波快速测量造球预配矿含水量的新方法,并详细地分析和讨论不同造球预配矿含水量对检测结果的影响.实验结果表明,当造球预配矿含水量为7.06 %～8.93 %时,检测误差小于0.28 %.这表明用微波谐振腔法快速测量造球预配矿含水量的方法是可行的.     

Applications of the PM3 semi-empirical method to the study of triethylenediamine

Charcoal filters impregnated with triethylenediamine (TEDA) are known to be efficient for the collection of volatile methyl iodide, which may be released under a hypothetical loss-of-coolant accident in a nuclear generating station. The structure and thermodynamic stability of the products of the TEDA-methyl iodide reaction have thus been studied using semi-empirical techniques. The reaction of TEDA with two molecules of methyl iodide leads to a quaternization reaction at each of the nitrogens. Moreover, it is shown that substitution of the hydrogens on TEDA with electron-donating groups can lead to enhanced stability of the quaternary ammonium reaction products. The semi-empirical method PM3 (Parametric Method 3) was used as the basis for all calculations. Molecular systems and simulations were constructed using HyperChem 4.5 for Silicon Graphics workstations. Enthalpy determination and geometry optimization were some of the calculations performed on a system.     

Use of thermal fluctuations to study the length and flexibility of ligand-receptor bonds

We describe an experimental approach yielding new information on the behavior of ligand-receptor bonds. Spherical particles of 1.4 microns radius were coated with anti-rabbit immunoglobulin monoclonal antibodies and deposited on surfaces derivatized with rabbit immunoglobulins. Brownian motion was studied. When particles where bound through multiple bonds, the mean square displacement during a 1 s interval was 0.0038 micron2 as compared to 0.245 micron2 for unbound particles. Under the same conditions, the mean square displacement of particules coated with limiting dilutions of binding sites and bound by single molecular bonds was 0.0774 micron2. Results are compatible with the concept that the latter particles behaved as spheres transiently bound to the substratum by links of 2.7 nm length, allowing brownian oscillations with an angular amplitude of 0.062 radian.     

Use of FLP/FRT system to study Drosophila development

Mycoplasma fermentans and other Mycoplasma species are colonizers of human mucosal surfaces and may be associated with human immunodeficiency virus infection. While many infectious agents have been described in different percentages of patients with Chronic Fatigue Syndrome (CFS), little is known about the prevalence of mycoplasmas and especially M. fermentans in CFS patients. A polymerase chain reaction (PCR)-based assay was used to detect Mycoplasma genus and M. fermentans genomes in peripheral blood mononuclear cells (PBMC) of CFS patients. Blood was collected from 100 patients with CFS and 50 control subjects. The amplified products of 717 bp of Mycoplasma genus, and 206 bp of M. fermentans were detected in DNA purified from blood samples in 52% and 34% of CFS samples, respectively. In contrast, these genomes were found in only 14% and 8% of healthy control subjects respectively (P < 0.0001). All samples were confirmed by Southern blot with a specific probe based on internal sequences of the expected amplification product. Several samples, which were positive for Mycoplasma genus, were negative for M. fermentans indicating that other Mycoplasma species are involved. A quantitative PCR was developed to determine the number of M. fermentans genome copies present in 1 microg of DNA for controls and CFS patients. Mycoplasma copy numbers ranging from 130 to 880 and from 264 to 2400 were detected in controls and CFS positive subjects, respectively. An enzyme immunoassay was applied for the detection of antibodies against p29 surface lipoprotein of M. fermentans to determine the relationship between M. fermentans genome copy numbers and antibody levels. Individuals with high genome copy numbers exhibited higher IgG and IgM antibodies against M. fermentans specific peptides. Isolation of this organism by culture from clinical specimens is needed in order to demonstrate specificity of signal detected by PCR in this study.     

Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms

Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates having substitutions at only one or two positions. We examined Kex2 sequence recognition for residues at P3, P2, and P1 using two types of fluorogenic peptide substrates, peptidyl-methylcoumarinamides and internally quenched substrates in which cleavage occurs at an actual peptide bond. Kinetic analysis of the two sets of substrates gave comparable data on specificity at these three positions. For the best substrate sequences, high catalytic constants (kCM/KM) of (2-5) x 10(7) M-1 s-1 were seen for cleavage of both peptidyl-methylcoumarinamides and peptide bonds. While no evidence for positive interactions with the P3 residue emerged, Kex2 was found to discriminate against at least one residue Asp. at this position. Specificity at P2 was shown to rely primarily on recognition of a positive charge, although steric constraints on the P2 side chain were also apparent. Kex2 was demonstrated to be exquisitely selective for Arg at P1. Substitutions with similar charge (Lys, ornithine) or similar hydrogen-bonding capability (citrulline) do not confer efficient catalysis. Comparison of otherwise identical substrates having either Arg or citrulline at P1 showed that the positive charge of the Arg guanidinium group stabilizes the transition state by approximately 6.8 kcal/mol.     

Sequence variation of the tRNA(Leu) intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA(Leu) (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA(Leu) (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA(Leu) (UAA) intron can be of great value when examining cyanobacterial diversity.     

Immunohistochemical study of mixed tumor of the skin with marked ossification

A seroepidemiological study was conducted in Kuwait to evaluate the pattern of acquisition of human parvovirus B19 by children in Kuwait and to compare it with patterns described in other regions in different climatic zones. A total of 218 serum samples from children less than 16 years of age were tested for the presence of anti-B19 IgG by enzyme immunoassay. The overall seroprevalence was 17.4%. Infants in Kuwait had low levels of maternally-acquired anti-B19 IgG (8.6%). The age of peak exposure to parvovirus B19 was 10-15 years compared with less than 10 years in England and Wales and more than 20 years in Singapore. The results of this study indicate an influence of geographic differences on transmission of parvovirus B19.