The role of Stn1p in Saccharomyces cerevisiae telomere capping can be separated from its interaction with Cdc13p

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.     

Human galectin‐3 interacts with two anticancer drugs

Human galectin‐3 (hGal‐3) is a mammalian lectin involved in regulation of RNA splicing, apoptosis, cell differentiation, and proliferation. Multimerized extracellular hGal‐3 is thought to crosslink cells by binding to glycoproteins and glycosylated cancer antigens on the cell surface or extracellular matrix. Fluorescence spectroscopy and circular dichroism were used to study the interaction of hGal‐3 with two anticancer agents: bohemine and Zn porphyrin (ZnTPPS₄). The dissociation constant (k_D) for binding of bohemine with hGal‐3 was k_D 0.23±0.05 μM. The hyperbolic titration curve indicated the presence of a single bohemine binding site. The binding of ZnTPPS₄ to hGal‐3 (with and without lactose) is of high affinity having k_D=0.18–0.20 μM and is not inhibited by lactose, indicating that ZnTPPS₄ and carbohydrate bind different sites. Circular dichroism spectra of the hGal‐3 complexes suggested that the binding of the hydrophobic compounds changed the hGal‐3 secondary structure. In summary, we show that two compounds with anticancer activity, bohemine and ZnTPPS₄, have high affinity for hGal‐3 at a site that is distinct from its carbohydrate site. Since hGal‐3 binds to several carbohydrate cancer antigens, the results suggest that it may have utility in the targeted delivery of drugs for cancer.     

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.     

Micro Cell Culture Method for Isolation of Chlamydia trachomatis

Presterilized mictotiter plates (96 wells) with BHK-21 cells on 5-mm cover slips were successfully used for cell culture isolation of trachoma from 15 infected conjunctival scrapings.     

Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.     

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 2-ethyl-5-methyl-3,3-diphenylpyraline and methadol in meconium by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

This paper details a validated liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method for the quantification of methadone, and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) and methadol in human meconium. Limits of detection (LOD) were determined to be 1.0 ng/g for methadone, EDDP and EMDP and 2.5 ng/g for methadol. The limits of quantitation (LOQ) for methadone, EDDP, EMDP were 5 and 25 ng/g for methadol. Linearity ranged from 5.0 to 500 ng/g. Following solid-phase extraction, no matrix effect was observed. This method proved to be suitable for the quantification of methadone, EDDP and EMDP and the semi-quantitation of methadol in meconium. Literature review revealed no other published LC-APCI-MS/MS method for the detection of methadone and its three main metabolites in meconium specimens.