The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.     

Secondary metabolites from Centaurea deusta with antimicrobial activity

The aerial parts of Centaurea deusta Ten. afforded in addition to several known compounds, mainly sesquiterpene lactones, one new eudesmanolide and one new elemane derivative. Structures of the new compounds were elucidated by spectroscopic methods. The in vitro antifungal and antibacterial activities of the isolated compounds was tested, using the microdilution method. All compounds tested showed high antifungal activity.     

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.     

Simple and rapid physico-chemical methods to examine action of antimalarial drugs with hemin: its application to Artemisia annua constituents

Malaria is a major health problem in many countries and according to an estimate of the WHO, more than 500 million infections occur per year. Artemisinin, a sesquiterpene from Artemisia annua L., has received considerable attention as a promising and potent antimalarial drug for its stage speciticity, its rather low toxicity, effectiveness against drug-resistant Plasmodium species and activity against cerebral malaria. From recent studies it seems that hemin is primarily involved in the antimalarial activity of the constituents of Artemisia annua L. Thus, the interaction of a compound with hemin may represent a crucial screening test to define its efficacy. In this study the interaction between artemisinin and hemin was investigated by UltraViolet/Visible (UV/Vis) spectrophotometry and High Performance Liquid Chromatography/Diode Array Detector/Mass Spectrometry (HPLC/DAD/MS). In addition, some flavonols isolated from Artemisia annua L. were also tested to investigate their possible role in the interaction between artemisinin and hemin. These two simple physico-chemical methods can be useful as rapid and widespread screening methods for the search of other alkylating antimalarial constituents from natural sources or for the evaluation of the activity of semisynthetic analogues of artemisinin.     

Ku antigen,an origin-specific binding protein that associates with replication proteins,is required for mammalian DNA replication

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.     

Chemical Diversity of Wild Growing Origanum majorana in Cyprus

The present work aims at tracing the essential‐oil diversity of wild growing Origanum majorana plants in Cyprus. The study of six populations scattered over the western part of the island has shown that the essential oils were rich either in trans‐sabinene hydrate/terpinen‐4‐ol or in α‐terpineol/trans‐sabinene hydrate. The former oil type is more common and responsible for the typical marjoram odor of the plants. The comparison of our results with published data concerning wild and cultivated O. majorana showed that the oil composition of the Cypriot populations had not been reported from wild plants from other parts of the Mediterranean region, while it is very common among the cultivated plants. Thus, it seems probable that the nowadays cultivated marjoram may originate from Cypriot wild populations.     

Effects of chilling stress on leaf morphology,anatomy, ultrastructure,gas exchange,and essential oils in the seasonally dimorphic plant Teucrium polium (Lamiaceae)

The Mediterranean region (and globally also other regions) is characterized by the presence of phryganic plants, i.e. subshrubs that grow under hot and arid environmental conditions. These plants are reported to be affected by summer drought stress. However, in the present study the phryganic plant Teucrium polium (mountain germander) appears to be affected by winter chilling stress rather than by summer drought stress in a specific area. Winter leaves of the plant are smaller and thicker compared to summer leaves, have more stomata and glandular hairs, and their chloroplasts are larger, more numerous, with voluminous starch grains. Moreover, epidermal and mesophyll cells of winter leaves contain in their vacuoles dark phenolics and calcium oxalate crystals. Summer leaves are devoid of vacuolar phenolics and their chloroplasts possess many large plastoglobuli. Leaf gas exchange parameters (photosynthesis, transpiration, stomatal conductance) are significantly higher in winter leaves. Concentrations of osmoprotectors (stress indicators) like proline and soluble sugars are similarly higher in winter leaves. Essential oil assessments showed a significantly higher oil yield of winter leaves compared to summer leaves. Percentages of the major oil components (linalool, terpinen-4-ol, germacrene D, and spathulenol) are remarkably higher in winter oils than in summer oils. In conclusion, low environmental temperatures (1–10 °C) appear to decisively influence the structure and function of winter leaves compared to summer leaves. Winter plants undergo chilling stress to which they respond by developing various mechanical and chemical defensive strategies.     

Essential Oils of Micromeria dalmatica Benth., a Balkan Endemic Species of Section Pseudomelissa

The essential oils of 13 Greek populations of Micromeria dalmatica, a Balkan endemic species and member of the section Pseudomelissa, were examined for the first time. Among the studied populations, two main oil types could be distinguished. Type I was found to be rich in β‐pinene, limonene, and germacrene D (accounting for 55.6–70.2% of the total oil), and Type II was characterized by the preponderance of p‐menthane compounds (accounting for 64.2–89.9% of the oil). The latter oil type could be further divided into two subtypes, one comprising oils with predominance of piperitenone and piperitenone oxide and another composed of oils containing high proportions of pulegone, menthone, and isomenthone. The abundance of p‐menthane compounds is a common feature of the oils of all members of the section Pseudomelissa studied to date. However, the existence of oils of Type I has not been previously reported for M. dalmatica, neither for other members of the section Pseudomelissa.