Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.     

The interval shuttle run test for intermittent sport players: evaluation of reliability

The reliability of the interval shuttle run test (ISRT) as a submaximal and maximal field test to measure intermittent endurance capacity was examined. During the ISRT, participants alternately run for 30 seconds and walk for 15 seconds. The running speed is increased from 10 km.h(-1) every 90 seconds until exhaustion. Within a 2-week period, 17 intermittent sport players (i.e., 10 men and 7 women) performed the ISRT twice in a sports hall under well-standardized conditions. Heart rates per speed and total number of runs were assessed as submaximal and maximal performance measures. With the exception of the heart rates at 10.0 km.h(-1) for men and 10.0, 12.0, and 13.5 km.h(-1) for women, zero lay within the 95% confidence interval of the mean differences, indicating that no bias existed between the outcome measures at the 2 test sessions (absolute reliability). The results illustrate that it is important to control for heart rate before the start of the ISRT. Relative reliability was high (intraclass correlation coefficient > or = 0.86). We conclude that the reliability of the ISRT as a submaximal and maximal field test for intermittent sport players is supported by the results.     

Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0A crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel alpha2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to "breathe" and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.     

Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.     

In vitro selection of second site revertants analysis of the hairpin ribozyme active site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study

BACKGROUND: The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary. RESULTS: We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm). CONCLUSIONS: Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.     

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.