Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.     

A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography

A systematic method for the sensitive, precise and accurate determination of hair lipids, including trace amounts of intrinsic endogenous cholesterol (CH), ceramide/N-palmitoyl-DL-dihydrosphingosine (CER/PDS), cholesterol sulfate (CS) and chemically bound 18-methyl eicosanoic acid (18-MEA), has been developed in combination with TLC/FID (flame ionization detection), LC/MS and GC/MS. TLC/FID was used for the simultaneous determination of squalene (SQ), wax esters (WEs), triglycerides (TGs) and free fatty acids (FFAs). Optimal conditions for LC/MS to determine CS and 18-MEA were developed using selected ion monitoring (SIM) under the negative ion mode of electrospray ionization. An alternative procedure for the determination of 18-MEA was also established using commercially available heneicosanoic acid (HEA). In GC/MS, the optimal selection of ions for SIM of trimethylsilylated CH and CER/PDS, and the use of on-column injection has enabled their simultaneous detection. This newly developed method has been used to characterize the hair lipid composition from the proximal root end to the distal tip of chemically untreated hair fibers from two different females, and specific changes of hair lipids probably due to its origin and individuals have been demonstrated for the first time. This method may be useful for clarifying the important roles of intrinsic endogenous 18-MEA, CS, CH and CERs in the function of the cell membrane complex of hair fibers.     

A critical role for thrombin in vertebrate lens regeneration

Lens regeneration in urodele amphibians such as the newt proceeds from the dorsal margin of the iris where pigment epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. A general problem in regeneration research is to understand how the events of tissue injury or removal are coupled to the activation of plasticity in residual differentiated cells or stem cells. Thrombin, a pivotal regulator of the injury response, has been implicated as a regulator of cell cycle re-entry in newt myotubes, and also in newt iris PEC. After removal of the lens, thrombin was activated on the dorsal margin for 5-7 days. Inactivation of thrombin by either of two different inhibitors essentially blocked S-phase re-entry by PEC at this location. The axolotl, a related species which can regenerate its limb but not its lens, can activate thrombin after amputation but not after lens removal. These data support the hypothesis that thrombin is a critical signal linking injury to regeneration, and offer a new perspective on the evolutionary and phylogenetic questions about regeneration.     

Synthesis and preliminary biological evaluation of truncated zoanthenol analogues

Zoanthamines are a family of marine alkaloids that have complex heptacyclic structures and are reported to be interleukin-6 modulators. While the structure of zoanthamines, especially the ABC-ring portion, is similar to that of steroids, the CDEFG-ring portion, composed of aminoacetal and lactone core, is a unique structural element. In this report, we designed and synthesized ABC-ring 6 and CDEFG-ring 7, which are truncated analogues of the northern and southern hemispheres of zoanthenol 5, respectively, and which incorporate all of the functionality of each hemisphere. A preliminary SAR study suggested that the hydrochloride of the CEFG-ring portion is an active pharmacophore for suppressing the growth of interleukin-6-dependent MH60 cells.     

Lanostane triterpenoids from the inedible mushroom Fomitopsis spraguei

Investigation of the methanolic extract of the inedible mushroom Fomitopsis spraguei (Polyporaceae) led to the isolation of five lanostane-type triterpenoids (1-5): three new compounds named fomitopsins A-C (2-4), and two known compounds, quercinic acid C (1) and 3alpha-carboxyacetyl-12beta-hydroxyquercinic acid (5). Their structures were determined by 2D NMR, MS, IR, UV spectra, and X-ray crystallographic analyses. An X-ray crystal structure analysis of quercinic acid C (1) established its stereochemistry as 3R,12R-dihydroxy-24R-methyl-23-oxo-25S-lanost-8-en-26-oic acid.     

Reorganized actin filaments anchor chloroplasts along the anticlinal walls of <Emphasis Type="Italic">Vallisneria</Emphasis> epidermal cells under high-intensity blue light

In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15–20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells.     

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.