Tetrahymena fimbrin localized in the division furrow bundles actin filaments in a calcium-independent manner

In cytokinesis, the contractile ring constricts the cleavage furrow. However, the formation and properties of the contractile ring are poorly understood. Fimbrin has two actin-binding domains and two EF-hand Ca(2+)-binding motifs. Ca(2+) binding to the EF-hand motifs inhibits actin-binding activity. In Tetrahymena, fimbrin is localized in the cleavage furrow during cytokinesis. In a previous study, Tetrahymena fimbrin was purified with an F-actin affinity column. However, the purified Tetrahymena fimbrin was broken in to a 60 kDa fragment of a 70 kDa full length fimbrin. In this study, we investigated the properties of recombinant Tetrahymena fimbrin. In an F-actin cosedimentation assay, Tetrahymena fimbrin bound to F-actin and bundled it in a Ca(2+)-independent manner, with a K(d) of 0.3 micro M and a stoichiometry at saturation of 1:1.4 (Tetrahymena fimbrin: actin). In the presence of 1 molecule of Tetrahymena fimbrin to 7 molecules of actin, F-actin was bundled. Immunofluorecence microscopy showed that a dotted line of Tetrahymena fimbrin along the cleavage furrow formed a ring structure. The properties and localization of Tetrahymena fimbrin suggest that it bundles actin filaments in the cleavage furrow and plays an important role in contractile ring formation during cytokinesis.     

Insertion of the membrane-proximal region of the neuronal SNARE coiled coil into the membrane

In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an alpha-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.     

Pimarane cyclooxygenase 2 (COX-2) inhibitor and its structure-activity relationship

The structure-activity relationship and molecular modelings of a novel pimarane COX-2 inhibitor are reported. Particularly, a series of linker extended analogues designed on the basis of these studies exhibited significantly enhanced COX-2 inhibitory activities and selectivities.     

A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus

Hot pepper (Capsicum annuum) plants exhibit a hypersensitive response (HR) against infection by many tobamoviruses. A clone (CaPR-4) encoding a putative pathogenesis-related protein 4 was isolated by differential screening of a cDNA library prepared from resistant pepper plant leaves inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaPR-4 is very similar to those of other plant PR-4s. Southern blot analysis showed that small gene families of PR-4-related sequences were present in the pepper genome. Hot pepper cultivar Bugang, resistant to TMV-P0 and susceptible to TMV-P1.2, induced CaPR-4 expression by pathotype P0 inoculation in inoculated and systemic leaves, but not by pathotype P1.2. Effects of exogenously applied abiotic elicitors upon the CaPR-4 expression were also examined. The expression of the CaPR-4 gene was stimulated by methyl jasmonate (MeJA), ethephon and wounding treatment. However, application of salicylic acid (SA) did not trigger the expression. Evidence is emerging that jasmonic acid and ethylene play key roles in the SA-independent pathways of plant-pathogen interaction. Taken together, these results suggest that the CaPR-4 gene is one of the defense-related genes conferring resistance on pepper plants by the SA-independent pathway and the cross-talk between signaling compounds, jasmonic acid and ethylene could have a great regulatory potential in a plant's defense against TMV.     

Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.     

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.     

Apoptosis-inducing protein, AIP, from parasite-infected fish induces apoptosis in mammalian cells by two different molecular mechanisms

AIP (apoptosis-inducing protein) is a protein purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, which induces apoptosis in various mammalian cells including human tumor cell lines. AIP has shown structural and functional homology to L-amino acid oxidase (LAO) which oxidizes several L-amino acids including L-lysine and AIP-induced apoptosis has been suggested to be mediated by H2O2 generated by LAO activity of AIP. In this study, we confirmed that recombinant AIP generated enough H2O2 in culture medium to induce rapid apoptosis in cells and this apoptosis was clearly inhibited by co-cultivation with antioxidants such as catalase and N-acetyl-cysteine. Surprisingly, however, we found that AIP still could induce H2O2-independent apoptosis more slowly than H2O2-dependent one in HL-60 cells even in the presence of antioxidants. In addition, the HL-60-derived cell line HP100-1, which is a H2O2-resistant variant, underwent apoptosis on treatment with AIP with a similar delayed time course. The latter apoptosis was completely blocked by addition of L-lysine to the culture medium, which is the best substrate of AIP as LAO, indicating that decreased concentration of L-lysine in the culture medium by AIP-treatment induced apoptosis. We also showed that the both apoptosis by AIP were associated with the release of cytochrome c from mitochondria and activation of caspase-9, and overexpressed Bcl-2 could inhibit both of the AIP-induced apoptosis. These results indicate that AIP induces apoptosis in cells by two distinct mechanisms; one rapid and mediated by H2O2, the other delayed and mediated by deprivation of L-lysine, both of which utilize caspase-9/cytochrome c system.     

Lamivudine production via enantioselective deamination by thermostable Bacillus caldolyticus cytidine deaminase

To decrease the costs of producing the anti-HIV drug, lamivudine, an enzymatic conversion process was developed instead of the traditional chemical method. Thermostable cytidine deaminase was over-produced by cloning the cdd gene into E. coli JF611/pCJH53 from Bacillus caldolyticus. The purified cytidine deaminase was recovered from the lysate of the recombinant E. coli JF611/pCJH53 by removing heat-denatured proteins and eluting sequential chromatography. When the enzyme was used to deaminate (–)--l-(2R, 5S)- and (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine, about 68% of the (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine was deaminated into the corresponding (+)-thiauridine maximally.     

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system

AIMS: The lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS. METHODS AND RESULTS: Exposure of E. coli to the LPAS, upon which 99.6% of the bacteria were killed, resulted in the following percentage of inactivation of substrate-specific membrane oxidases: succinate (94.2%) > NADH (84.6%) > glycerol-3-phosphate (67.8%) > DL-lactate (64.1%). With the same treatment, substrate-specific membrane dehydrogenases were inactivated as follows: succinate (99.1%) > DL-lactate (53.8%) > glycerol-3-phosphate (45.0%) > NADH (36.8%). Terminal oxidase, however, measured using a ubiquinone analogue (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) after reduction, was only 21.4% inactivated by the LPAS. CONCLUSION: These data suggest that dehydrogenases are the primary targets of the LPAS in the respiratory chain of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has determined for the first time the primary targets of LPAS in the bacterial respiratory chain.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.