Molecular and genetic studies of fusarium trichothecene biosynthesis: pathways, genes, and evolution

Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.     

Effects of initial settings on computational protein–ligand docking accuracies for several docking programs

In this study, the influences of initial settings, i.e. initial conformations, configurations and docking parameters, on docking results were investigated. The conformations used in the study were generated by the CAMDAS program. After the conformational search calculations, five structures were selected from the conformer groups according to their conformation energies and root mean square deviations against crystal structures; for example, the lowest energy conformer, as well as the closest and farthest conformers to the crystal structure, was retrieved. Several docking parameter settings were used (default, high speed, generating 50 poses). In this study, docking calculations were conducted using the GOLD, eHiTS, AutoDock, AutoDock vina, FRED and DOCK programs. The success rates of GOLD, eHiTS and FRED were better than those of AutoDock, AutoDock vina and DOCK. The docking results using the farthest conformations were worse than those obtained using other conformations, indicating that some conformation search for the ligand molecule should be performed before the docking calculations.     

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.     

Chromosome analysis by spectral karyotyping of spermatozoa from an oligoasthenozoospermic carrier of a 10; 21 reciprocal translocation

Cytogenetic analysis of germ-line cells prior to intracytoplasmic sperm injection (ICSI) treatment is thought to be necessary for infertile males with an identified chromosomal abnormality. We analyzed the chromosomal karyotype of human spermatozoa from an oligoasthenozoospermic carrier of a reciprocal translocation t(10; 21). Cytogenetic analysis of 39 spermatozoa was performed by spectral karyotyping (SKY) and by ICSI into mouse oocytes. The motile morphologically normal spermatozoa were injected into mouse oocytes. Of these spermatozoa, 38 (97.4%) were activated. Twenty-one (53.8%) of the activated oocytes formed two pronuclei. Metaphase chromosome spreads from 13 spermatozoa were analyzed. Only one spermatozoon was normal and 2 spermatozoa exhibited balanced translocation. Nine and one spermatozoa showed abnormalities related and unrelated to the translocation, respectively. The numbers of normal/balanced spermatozoa were lower than those in previous reports analyzing reciprocal translocations using a previously described technique involving penetrated golden hamster oocytes. After genetic counseling with the carrier and his partner, ICSI treatment was performed. Healthy female and male infants were delivered at 37 weeks gestation via a Caesarean section. The female infant was a carrier of the reciprocal translocation and the male infant was confirmed normal on prenatal diagnosis at 16 weeks gestation. For genetic counseling prior to ICSI treatment, the incidence of unbalanced type spermatozoa after swim-up or Percoll gradient treatment should be investigated and discussed with couples having fertility problems related to oligozoospermia autosomal structural abnormalities.     

Estimating intercellular surface tension by laser-induced cell fusion

Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 μN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.