In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.     

The nutritional contribution of males affects the feeding behavior and spatial distribution of females in a bruchid beetle,<Emphasis Type="Italic"> Bruchidius dorsalis</Emphasis>

The relationship between the quality/quantity of male investment and the feeding behavior of females was investigated in a bruchid weevil, Bruchidius dorsalis (Fahraeus), whose males donate nutrition via seminal fluid to females. Experiments on the effect of feeding regimes of both sexes on the mating frequency of females showed that females mated at a higher frequency if given low-quality food or poor male investment. On the other hand, the experiment that examined the effect of male investment quality on female feeding behavior showed that females receiving the high-quality investment exhibited feeding behavior less often. These results suggest that male investment and feeding behavior play the same role for B. dorsalis females. These experiments also showed that there are sex-related asymmetries in mating and feeding behaviors: females mated more often but males fed more often. Moreover, a field census suggested that only males visited non-host flowers to feed on the pollen and nectar during the non-flowering period of the host plants; females always stayed on the host plants irrespective of the flowering phenology. These results suggest that in B. dorsalis courtship role reversal and sex-specific feeding modes are fairly fixed and obligatory, and that male investment, derived from sexual selection, could affect the feeding behavior and spatial distribution of both sexes, which may have far-reaching impact in various ecological contexts.     

Phylogenetic Analysis of Isolates of Pasteurella pneumotropica from Laboratory Animals Based on the gyrB Gene Sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.     

Reproductive interference between two serious pests,oriental fruit flies <Emphasis Type="Italic">Bactrocera carambolae</Emphasis> and <Emphasis Type="Italic">B. dorsalis</Emphasis> (Diptera: Tephritidae), with very wide but partially overlapping host ranges

Bactrocera carambolae and B. dorsalis (Diptera: Tephritidae) are extremely destructive pests of fruits and vegetables in the Asia-Pacific region. Earlier reports have described that B. carambolae and B. dorsalis, respectively, use mainly star fruit and mango, suggesting a certain level of host partitioning which can be ascribed neither to differences in larval food qualities nor host-specific parasitoid mortality. This study specifically examined reproductive interference (antagonistic sexual interaction) between B. carambolae and B. dorsalis as a potential factor strongly affecting their host partitioning. We observed mating behaviors, especially interspecific courtships and mating, by cohabiting the conspecific and heterospecific pairs together. Consequently, we quantified their effects on the reproductive success of females. Males of both species frequently courted their own females, but they also courted females of other species. Courtship refusal by females was not selective in males of either species. This incomplete discrimination of both sexes led to frequent occurrences of interspecific sexual interactions in both species, but only B. carambolae females showed reduced reproductive success. These results suggest that B. dorsalis, superior in reproductive interference, can occupy high-quality mango, whereas B. carambolae, inferior in reproductive interference, must use low-quality star fruit.     

Active female courtship behavior and male nutritional contribution to female fecundity in Bruchidius dorsalis (Fahraeus) (Coleoptera: Bruchidae)

Mating behavior and the male's contribution to female fecundity were studied in the bean weevil Bruchidius dorsalis (Fahraeus) (Coleoptera: Bruchidae) in comparison with two other species, Callosobruchus chinensis (which infests stored beans) and Kytorhinus sharpianus (which feeds on wild legumes). Only females of B. dorsalis showed multiple mating and characteristic precopulatory behavior that appeared to solicit the male's nutritious secretion. In contrast, all females of the other two species did not copulate multiply and did not show such precopulatory behavior. In B. dorsalis, the decrement of male body weight just after copulation indicated that seminal fluid weighing as much as approximately 7% of the male's body weight was transferred to the female. Fecundity was more than eight times higher in females that had copulated ten times than in females that had copulated only once, indicating that males paid most of the nutritional cost of egg production. These facts suggest that the sex role is reversed in B. dorsalis. Received: May 22, 1998 / Accepted: July 19, 1999     

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of 相似文献   

Development of α1,6-fucosyltransferase inhibitors through the diversity-oriented syntheses of GDP-fucose mimics using the coupling between alkyne and sulfonyl azide

We developed α1,6-fucosyltransferase (FUT8) inhibitors through a diversity-oriented synthesis. The coupling reaction between the fucose unit containing alkyne and the guanine unit containing sulfonyl azide under various conditions afforded a series of Guanosine 5′-diphospho-β-l-fucose (GDP-fucose) analogs. The synthesized compounds displayed FUT8 inhibition activity. A docking study revealed that the binding mode of the inhibitor synthesized with FUT8 was similar to that of GDP-fucose.