IGF-1 Participates Differently in Regulation of Severing Activity of Katanin and Spastin

Spastin and p60-katanin are AAA family proteins that participate in microtubule severing, while lipotransin, another AAA family protein is a hormone sensitive lipase interacting protein. Sequence alignment analysis suggests that lipotransin and human p60-katanin are the orthologs of each other. Studies identified that insulin may negatively regulate ATPase activity of lipotransin. To reveal the effects of insulin on regulation of severing activity of p60-katanin and spastin, hippocampal neurons over-expressing spastin and p60-katanin were treated with IGF-1. Changes in neuronal branching by considering the total process lengths and average process numbers were quantitatively analyzed. According to the results of this study, total process lengths of hippocampal neurons and average process numbers remained similar in control and p60-katanin over-expressing neurons upon IGF-1 treatment, while significant decrease was observed in spastin over-expressing neurons. This study indicated that IGF-1 participates differently in the regulation of spastin and p60-katanin in terms of neuronal branching.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.     

The apoptotic effects of mitomycin C on human endometrial cell cultures and reversal of its effects by beta-carotene and folic acid

Apoptosis is a complex process involving a variety of mechanisms and it has been shown to be a response of cells to various chemical agents including chemotherapeutic ones. We aimed to induce DNA breaks and apoptosis in cultured endometrial stromal cells by mitomycin C (MMC), a chemotherapeutic agent, and also we aimed to observe the effects of beta-carotene and folic acid on MMC-induced apoptosis. Cultured endometrial stromal cells were exposed to MMC for 48 and 72 hours and in order to reverse MMC effects, we added beta-carotene and folic acid to the cultures. DNA fragmentation was observed in all cells. Apoptotic cell ratios and caspase-3 activity were observed to be dependent on exposure time. Ultrastructural examinations revealed positive effects of beta-carotene and folic acid, however they were not sufficient enough to prevent apoptosis in all cells. Beta-carotene profoundy reduced caspase-3 activity whereas folic acid did not seem to have a similar effect. As apoptosis involves several mechanisms, in a cell in which all these mechanisms are triggered, we think that antioxidants and DNA repair agents alone are not enough to reverse all of them.     

A karyological study of Asphodelus L. (Asphodelaceae) from the Western Mediterranean

The following aspects of Asphodelus karyology are analysed: base number, polyploidy, chromosome size, chromosome morphology, satellited chromosomes, structural heteromorphism, karyotype asymmetry and karyotype evolution. The base number 0 ×= 14 is common to all species except for A. refractus , which has the derived ×= 13. Three ploidy levels occur, often in the same species; diploid, tetraploid and hexaploid, with 2n = 28, 56 and 84. Chromosomes are generally small to medium-small, with the occasional presence of medium-large chromosomes. The most frequent chromosome types are metacentric of type m and submetacentric. Metacentric chromosomes of type M occur only in sections. Verineopsis, Verinea and Plagiasphodelus ; subtelocentric chromosomes occur only in sections Asphodelus and Plagiasphodelus. There is a wide variability in relation to the number of satellited chromosomes, relative to ploidy level. There are usually two to four in diploids, four to eight in tetraploids and usually six, exceptionally up to 12, in the hexaploid. Satellites are present on the shortest arm, exceptionally on the longest arm. There is a high degree of structural heteromorphism in practically all the species which affects satellited and non satellited chromosomes. Karyotype asymmetry is generally of type 2B. Inter-and intra-chromosomal differences are estimated by the A1 and A2 indexes. Both indices vary in the karyotype evolution of the genus, with a decrease of A1 and an increase of A2. The role of polyploidy, hybridization, asymmetry and decrease of chromosome size in the evolution of Asphodelus is discussed.     

POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.