Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Genetic differentiation, structure, and a transition zone among populations of the pitcher plant moth Exyra semicrocea: implications for conservation

Pitcher plant bogs, or carnivorous plant wetlands, have experienced extensive habitat loss and fragmentation throughout the southeastern United States Coastal Plain, resulting in an estimated reduction to <3% of their former range. This situation has lead to increased management attention of these habitats and their carnivorous plant species. However, conservation priorities focus primarily on the plants since little information currently exists on other community members, such as their endemic arthropod biota. Here, we investigated the population structure of one of these, the obligate pitcher plant moth Exyra semicrocea (Lepidoptera: Noctuidae), using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. Examination of 221 individuals from 11 populations across eight southeastern US states identified 51 unique haplotypes. These haplotypes belonged to one of two divergent (～1.9-3.0%) lineages separated by the Mississippi alluvial plain. Populations of the West Gulf Coastal Plain exhibited significant genetic structure, contrasting with similarly distanced populations east of the Mississippi alluvial plain. In the eastern portion of the Coastal Plain, an apparent transition zone exists between two regionally distinct population groups, with a well-established genetic discontinuity for other organisms coinciding with this zone. The structure of E. semicrocea appears to have been influenced by patchy pitcher plant bog habitats in the West Gulf Coastal Plain as well as impacts of Pleistocene interglacials on the Apalachicola-Chattahoochee-Flint River Basin. These findings, along with potential extirpation of E. semicrocea at four visited, but isolated, sites highlight the need to consider other endemic or associated community members when managing and restoring pitcher plant bog habitats.     

Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at -40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry-based techniques (e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry), NMR spectroscopy and enzymatic assays.     

Combined treatment of heterocyclic analogues and benznidazole upon Trypanosoma cruzi in vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via i.p. route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals.     

Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) -- bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.     

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22⁺ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.Abbreviations AcBut 4-(4-Acetylphenoxy) butanoic acid - AcPAc (3-Acetylphenyl) acetic acid - ATC Antibody-targeted chemotherapy - BCL B-cell lymphoma - CalichDM N-Acetyl--calicheamicin dimethyl disulfide derivative(s) - CalichDMA CalichDM acid - CalichDMH CalichDM hydrazide - CDR Complementarity determining region - NHL Non-Hodgkins lymphoma - PBMC Peripheral blood mononuclear cell - TAA Tumor-associated antigen     

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Antimicrobial peptides and endotoxin inhibit cytokine and nitric oxide release but amplify respiratory burst response in human and murine macrophages

Antimicrobial peptides (AMPs), in addition to their antibacterial properties, are also chemotactic and signalling molecules that connect the innate and adaptive immune responses. The role of AMP [alpha defensins, LL-37, a cathepsin G-derived peptide (CG117-136), protegrins (PG-1), polymyxin B (PMX) and LLP1] in modulating the respiratory burst response in human and murine macrophages in the presence of bacterial endotoxin [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] was investigated. AMP were found to neutralize endotoxin induction of nitric oxide and TNFalpha release in macrophages in a dose-dependent manner. In contrast, macrophages primed overnight with AMP and LOS or LPS significantly enhanced reactive oxygen species (ROS) release compared with cells primed with endotoxin or AMP alone, while no responses were seen in unprimed cells. This enhanced ROS release by macrophages was seen in all cell lines including those obtained from C3H/HeJ (TLR4-/-) mice. Similar effects were also seen when AMP and endotoxin were added directly with zymosan to trigger phagocytosis and the respiratory burst in unprimed RAW 264.7 and C3H/HeJ macrophages. Amplification of ROS release was also demonstrated in a cell-free system of xanthine and xanthine oxidase. Although AMP inhibited cytokine and nitric oxide induction by endotoxin in a TLR4-dependent manner, AMP and endotoxin amplified ROS release in a TLR4-independent manner possibly by exerting a prolonged catalytic effect on the ROS generating enzymes such as the NADPH-oxidase complex.     

Gene expression in the hippocampus of inbred alcohol-preferring and -nonpreferring rats

The hippocampus is sensitive to the effects of ethanol and appears to have a role in the development of alcohol tolerance. The objective of this study was to test the hypothesis that there are innate differences in gene expression in the hippocampus of inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats that may contribute to differences in sensitivity to ethanol and/or in the development of tolerance. Affymetrix microarrays were used to measure gene expression in the hippocampus of alcohol-naive male iP and iNP rats in two experiments (n=4 and 6 per strain in the two experiments). Combining data from the two experiments, there were 137 probesets representing 129 genes that significantly differed (P < or = 0.01); 62 probesets differed at P < or = 0.001. Among the 36% of the genes that were expressed more in the iP than iNP rat at this level of significance, many were involved in cell growth and adhesion, cellular stress reduction and anti-oxidation, protein trafficking, regulation of gene expression, synaptic function and metabolism. Among the 64% of the genes that had lower expression in the hippocampus of iP than iNP rats were genes involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death and neurotransmission. Overall, the data indicate that there are significant innate differences in gene expression in the hippocampus between iP and iNP rats, some of which might contribute to the differences observed in the development of alcohol tolerance between the selectively bred P and NP lines.