重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

Evolution of body condition‐dependent dispersal in metapopulations

Body condition‐dependent dispersal strategies are common in nature. Although it is obvious that environmental constraints may induce a positive relationship between body condition and dispersal, it is not clear whether positive body conditional dispersal strategies may evolve as a strategy in metapopulations. We have developed an individual‐based simulation model to investigate how body condition–dispersal reaction norms evolve in metapopulations that are characterized by different levels of environmental stochasticity and dispersal mortality. In the model, body condition is related to fecundity and determined either by environmental conditions during juvenile development (adult dispersal) or by those experienced by the mother (natal dispersal). Evolutionarily stable reaction norms strongly depend on metapopulation conditions: positive body condition dependency of dispersal evolved in metapopulation conditions with low levels of dispersal mortality and high levels of environmental stochasticity. Negative body condition‐dependent dispersal evolved in metapopulations with high dispersal mortality and low environmental stochasticity. The latter strategy is responsible for higher dispersal rates under kin competition when dispersal decisions are based on body condition reached at the adult life stage. The evolution of both positive and negative body condition‐dependent dispersal strategies is consequently likely in metapopulations and depends on the prevalent environmental conditions.     

Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin.

Mycocerosyl lipids are found uniquely in the cell walls of pathogenic mycobacteria. Mycocerosic acid synthase (MAS) is a multifunctional protein which catalyzes elongation of n-fatty acyl-CoA with methylamalonyl-CoA as the elongating agent (Rainwater, D. L., and Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 616-623). To understand how the various domains that catalyze the reactions involved in chain elongation are organized, mas gene from Mycobacterium tuberculosis bovis BCG was cloned. A lambda gt11 library of AluI partially digested genomic DNA from the organism was screened with an oligonucleotide probe designed from the N-terminal amino acid sequence of purified MAS. Using terminal segments of inserts from positive clones as the probe, the library was rescreened and the process was repeated. Sequencing of four overlapping clones revealed a contiguous sequence of 9699 base pair(s) (bp) of mycobacterial genome containing a 6330-bp open reading frame that could code for a protein of 2100 amino acids with a molecular mass of 225,437 daltons. The authenticity of the open reading frame as that of MAS was verified by correspondence of the amino acid sequences deduced from the gene with the directly determined amino acid sequences of the N terminus and three different internal peptide fragments. By comparing the MAS amino acid sequence with the sequences in the active site regions of known fatty acid synthases and polyketide synthases the functional domains in MAS were identified. This analysis showed that the domains were organized in the following order: beta-ketoacyl synthase, acyl transferase, dehydratase-enoyl reductase, beta-ketoreductase, acyl carrier protein; no thioesterase-like domain could be found. These results establish MAS as the first case of an elongating multifunctional enzyme composed of two identical subunits that resemble the vertebrate fatty acid synthase in size, subunit structure, and linear organization of functional domains. Southern and Western blot analyses showed absence of mas gene and encoded proteins in Mycobacterium smegmatis and Escherichia coli. This result is consistent with the report that mycocerosic acid is present only in pathogenic mycobacteria.     

Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides.

The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

Composition of suberin-associated waxes from the subterranean storage organs of seven plants

The waxes associated with the suberin in the periderm of the underground storage organs of parsnip (Pastinaca sativa L.), carrot (Daucus carota L.), rutabaga (Brassica napobrassica Mill.), turnip (Brassica rapa L.), red beet (Beta vulgaris L.), sweet potato (Ipomoea batatas L.) and potato (Solanum tuberosum L.) were isolated, fractionated into hydrocarbon, wax ester, free fatty alcohol and free fatty acid fractions, and analyzed by combined gas chromatography and mass spectrometry. The amount of wax extracted from the periderm of the storage organs ranged from 2 to 32 μg/cm². The hydrocarbons from the suberin layer have a broader chain-length distribution, a predominance of shorter carbon chains, and a higher proportion of even-numbered carbon chains than the leaf alkanes from the same plants. The major components of the free and esterified fatty alcohols and fatty acids have an even number of carbon atoms, and are similar in chain-length distribution to their counterparts found covalently attached to the suberin polymers; however, these suberin components are shorter in chain length than their cuticular analogues from the leaves. Also extracted from the storage organs were polar components which included fatty alcohols and fatty acids in a conjugated form, and ω-hydroxy acids and dicarboxylic acids. Evidence is presented that removal of the wax from the periderm of whole storage organs results in a decrease in diffusion resistance to moisture. Scientific Paper No. 5516, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Enzymatic esterification of alkane-2,3-diols by the microsomes of the uropygial glands of ring-necked pheasants (Phasianus colchicus)

Extracts of uropygial glands of ring-necked pheasants (Phasianus colchicus) catalyzed diester formation from tritiated C₁₈ alkane-2,3-diol. Monoacylated diol was also tentatively identified in the enzymatic product. Subcellular fractionation showed that the esterifying activity was located mainly in the microsomal fraction. ATP and CoA were required for the esterification process, and this reaction was stimulated by Mg²⁺. Source of the acyl moieties for esterification was endogenous, and addition of exogenous fatty acid inhibited the reaction. When microsomes were treated with bovine serum albumin (BSA) in order to remove the endogenous source of acyl moieties, palmitoyl-CoA substituted for the ATP and CoA requirement. The pH optimum with ATP and CoA was between 6.0–9.0, while maximal rates of esterification were obtained with palmitoyl-CoA from pH 7.0 to 9.0. Borate ions stimulated esterification. The half maximal velocity was obtained with 2.0 × 10^?4, m diol, and 7.2 × 10^?5, m palmitoyl-CoA. Thiol reagents severely inhibited the esterification reaction with ATP and CoA, while much less inhibition was observed when palmitoyl-CoA was used. It is concluded that a microsomal acyl-CoA-diol transacylase catalyzes stepwise acylation of alkane-2,3-diols to give the diol diester which constitute the major component of the uropygial lipids of ring-necked pheasants.     

Metabolism of Red Beet Slices II. Effects of Aging

The rate of respiration of red beet slices increased 3- to 4-fold when the slices were aged in a moist atmosphere for 18 to 24 hours. The respiration of fresh slices was severely inhibited by 8 × 10⁻⁵ m HCN but as slices aged the sensitivity of respiration to HCN fell rapidly. The presence of HCN stimulated the respiration of slices after 8 to 12 hours of aging.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.