Structural and functional properties of αβ-heterodimers of tropomyosin with myopathic mutations Q147P and K49del in the β-chain

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca²⁺-regulated contraction of striated muscles. Two Tpm isoforms, α (Tpm 1.1) and β (Tpm 2.2), are expressed in fast skeletal muscles. These Tpm isoforms can form either αα and ββ homodimers, or αβ heterodimers. However, only αα-Tpm and αβ-Tpm dimers are usually present in most of fast skeletal muscles, because ββ-homodimers are relatively unstable and cannot exist under physiologic conditions. Nevertheless, the most of previous studies of myopathy-causing mutations in the Tpm β-chains were performed on the ββ-homodimers. In the present work, we applied different methods to investigate the effects of two myopathic mutations in the β-chain, Q147P and K49del (i.e. deletion of Lys49), on structural and functional properties of Tpm αβ-heterodimers and to compare them with the properties of ββ-homodimers carrying these mutations in both β-chains. The results show that the properties of αβ-Tpm heterodimers with these mutations in the β-chain differ significantly from the properties of ββ-homodimers with the same substitutions in both β-chains. This indicates that the αβ-heterodimer is a more appropriate model for studying the effects of myopathic mutations in the β-chain of Tpm than the ββ-homodimer which virtually does not exist in human skeletal muscles.     

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Rat Small Intestine

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. I. Molecular, cellular and physiological characterization of the Arabidopsis bul1 mutant, defective in the Δ7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis

Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bul1-1 mutant indicated that bul1-1 is defective in the Δ⁷-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes. Received: 28 April 2000 / Accepted: 6 October 2000     

Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases

An insertion-sequence of prokaryotic origin was detected in a genomic clone obtained from a Phaseolus vulgaris bacterial artificial chromosome (BAC) library. This BAC clone, characterized as part of a contig constructed near a virus resistance gene, exhibited restriction fragment length polymorphism with an overlapping clone of the contig. Restriction analysis of DNA obtained from individual colonies of the stock culture indicated the presence of a mixed population of wild-type and insertional mutants. Sequence analysis of both members of the population revealed the presence of IS10R, an insertion-sequence from Escherichia coli. A BLAST search for IS10-like sequences detected unexpected homologies with a large number of eukaryotic sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogaster and Caenorhabditis elegans. Southern analysis of a random sample of BAC clones failed to detect IS10 in the BAC DNA. However, prolonged sub-culturing of a set of 15 clones resulted in transposition into the BAC DNA. Eventually, all cultures acquired a 2.3-kb fragment that hybridized strongly with IS10. Sequence analysis revealed the presence of a preferred site for transposition in the BAC vector. These results indicate that a large number, if not all, of the BAC libraries from different organisms are contaminated with IS10R. The source of this element has been identified as the DH10B strain of E. coli used as the host for BAC libraries. Received: 5 December 2000 / Accepted: 25 April 2001     

Expressed variants of Δ12-fatty acid desaturase for the high oleate trait in spanish market-type peanut lines

Increasing the oleic to linoleic acid ratio (O/L) in peanut has positiveeffects on peanut quality and its nutritional value. ¹²-Fattyacid desaturases (¹²-Fad) have been targeted as logicalcandidates controlling the high oleate trait. A previous study using genomicDNA identified an insertion and a polymorphism resulting in an amino acid changeassociated with the high oleate trait in Spanish-type peanut cultivars. Theobjectives of this research were to use RT-PCR to confirm that the SingleNucleotide Polymorphims (SNPs) identified by analysis of genomic DNA wereexpressed, and to determine if expression patterns for ¹²-Fadwere the same in both seeds and leaves. A polymorphic region of the¹²-Fad containing a series of nucleotide changes wasamplified, cloned, and sequenced from mRNA of 155 clones of two parental linesand their independent derived backcross lines (IDBLs). The latter differed intheir oleic to linoleic ratio. Data indicated that the Ainsertion and the amino acid change were expressed in both leaf and seed tissue of thehigh and low-intermediate O/L genotypes. It is postulated that several copiesof the ¹²-Fad are present in the genome. It is reasonable toconclude that total activity, and ultimately the O/L ratio, is dependent on thenumber of functional copies. The results provide the basis for an assay toscreen for the high O/L ratio at the molecular level. We also report thepresence of another isozyme of ¹²-Fad with high homology tosoybean isozyme 2 that was expressed in seeds. These authors contributed equally to this work     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

利用Tn5定位诱变筛选紫云英根瘤菌Exo^—变种

利用Ｔｎ５定位诱变方法,对质粒ｐＪＢＢ５进行Ｔｎ５插入诱变,得到１０个Ｔｎ５在５９ｋｂＢ５外源片段上有不同插入位点的质粒ＴＮ１１,ＴＮ１１２,ＴＮ２２,ＴＮ２３,ＴＮ３１,ＴＮ４１,ＴＮ９１,ＴＮ１０１,ＴＮ１３１,ＴＮ１４１。将Ｔｎ１１等分别转移到已经含有不相容质粒ｐＰＨ１ＪＩ的紫云英根瘤菌１０７菌株中,使之发生同源变换。通过抗性选择及表型鉴别,筛选到３株菌落表型干燥（Ｍｕｃ－）的酸性胞外多糖（ＥＰＳ）合成缺陷菌株（Ｅｘｏ－）１０７（ＴＮ２２）,１０７（ＴＮ１０１）,１０７（ＴＮ１３１）。Ｓｏｕｔｈｅｒｎ杂交分析证明这３株变种的Ｔｎ５插入确实是同源交换而不是转座产生,表明经过适当改良的Ｔｎ５定位诱变法可以应用于紫云英根瘤菌Ｅｘｏ－变种的筛选。     

Membranotropic effects of peptides from the V3 loop of HIV-1

Summary The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell-cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the ΔpH and the Δψ) of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an α-helix/β-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus-cell fusion.