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®.     

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.     

Autophagy and mTOR pathways in mouse embryonic stem cell,lung cancer and somatic fibroblast cell lines

Embryonic developmental stages and regulations have always been one of the most intriguing aspects of science. Since the cancer stem cell discovery, striking for cancer development and recurrence, embryonic stem cells and control mechanisms, as well as cancer cells and cancer stem cell control mechanisms become important research materials. It is necessary to reveal the similarities and differences between somatic and cancer cells which are formed of embryonic stem cells divisions and determinations. For this purpose, mouse embryonic stem cells (mESCs), mouse skin fibroblast cells (MSFs) and mouse lung squamous cancer cells (SqLCCs) were grown in vitro and the differences between these three cell lines signalling regulations of mechanistic target of rapamycin (mTOR) and autophagic pathways were demonstrated by immunofluorescence and real-time polymerase chain reaction. Expressional differences were clearly shown between embryonic, cancer and somatic cells that mESCs displayed higher expressional level of Atg10, Hdac1 and Cln3 which are related with autophagic regulation and Hsp4, Prkca, Rhoa and ribosomal S6 genes related with mTOR activity. LC3 and mTOR protein levels were lower in mESCs than MSFs. Thus, the mechanisms of embryonic stem cell regulation results in the formation of somatic tissues whereas that these cells may be the causative agents of cancer in any deterioration.     

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.     

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.     

Food resource matching by foraging tits Parus spp. during spring-summer in a Mediterranean mixed forest; evidence for an ideal free distribution

We analysed whether patterns of microhabitat use by Blue Tits Parus caeruleus , Great Tits Parus major and Crested Tits Parus cristatus inhabiting a mixed forest consistently matched the patterns of food availability experienced by foraging birds during spring-summer. The use of five microhabitats by each bird species (the foliage of three tree species, shrubs and ground) and the availability of food in trees during the prebreeding, breeding and post-breeding periods of the birds' annual cycle were measured. All three tit species foraged mainly in the outer part of tree canopies (small branches and leaves or needles). Tit distributions between tree species matched food resource distributions irrespective of overall food resource levels, which varied four-fold between the study periods, and tit species. Tits also exploited secondary microhabitats (shrubs and ground) in periods of low food availability; Blue Tits tended to use shrubs, whereas Great and Crested Tits foraged on the ground. Between-trees distributions fitted that expected from an ideal free distribution, suggesting that food availability and intraspecific exploitative competition were the main factors governing tree use by tits. In contrast, patterns of use of secondary microhabitats (shrubs and ground) seemed to indicate a role for the species-specific morphological configurations of each tit species since Blue Tits are better adapted to hang and tended to forage in shubs, whereas Great and Crested Tits are better adapted to feed on horizontal surfaces and tended to forage on the ground. No evidence of interspecific interactions was observed. Overall, the results pointed to an independent exploitation of Mediterranean mixed forest by each bird species, food availability and food accessibility being the main factors affecting microhabitat use by foraging tits.     

Factors Controlling the Abundance and Size Distribution of the Phototrophic Ciliate Myrionecta rubra in Open Waters of the North Atlantic

ABSTRACT. Myrionecta rubra, a ubiquitous planktonic ciliate, has received much attention due to its wide distribution, occurrence as a red tide organism, and unusual cryptophyte endosymbiont. Although well studied in coastal waters, M. rubra is poorly examined in the open ocean. In the Irminger Basin, North Atlantic, the abundance of M. rubra was 0–5 cells/ml, which is low compared with that found in coastal areas. Distinct patchiness (100 km) was revealed by geostatistical analysis. Multiple regression indicated there was little relationship between M. rubra abundance and a number of environmental factors, with the exception of temperature and phytoplankton biomass, which influenced abundance in the spring. We also improve on studies that indicate distinct size classes of M. rubra; we statistically recognise four significantly distinct width classes (5–16, 12–23, 18–27, 21–33 μm), which decrease in abundance with increasing size. A multinomial logistic regression revealed the main variable correlated with this size distribution was ambient nitrate concentration. Finally, we propose a hypothesis for the distribution of sizes, involving nutrients, feeding, and dividing of the endosymbiont.     

The regulatory effects of clomiphene and tamoxifen on mTOR and LC3-II expressions in relation to autophagy in experimental polycystic ovary syndrome (PCOS)

Molecular Biology Reports - Polycystic ovary syndrome (PCOS) is a metabolic disease that causes infertility due to anovulation in women in reproductive age. It is known that clomiphene citrate (CC)...     

In vitro activity of sodium benzoate against clinically relevant Enterococcus faecalis and Enterococcus faecium isolates

The antimicrobial effects of sodium benzoate against Enterococcus faecalis and Enterococcus faecium were investigated. The MIC(90) of sodium benzoate were 64 mg/L for E. faecalis and 32 mg/L for E. faecium, while the MBC(90) were 128 mg/L and 64 mg/L, respectively. Although further studies are required for clinical evidence, sodium benzoate seems to be effective against Enterococcus spp.