An increased receptive field of olfactory receptor Or43a in the antennal lobe of Drosophila reduces benzaldehyde-driven avoidance behavior

Most animals orient themselves in their environment through the perception of olfactory cues. In order to gain insight into the principles of olfactory processing in Drosophila, we misexpressed olfactory receptor Or43a in additional olfactory receptor neurons of the third antennal segment using enhancer trap line GH320. The behavioral response of GH320/UAS-or43a flies was changed upon benzaldehyde application. Using the T-maze assay, misexpressing flies performed a reduced avoidance reaction to benzaldehyde as compared with wild type. This reduction of avoidance could be mimicked in wild type flies by exposing them to a mixture of benzaldehyde and ethyl acetate. We therefore conclude that the application of benzaldehyde, an identified ligand of Or43a, resulted in activation of a number of glomeruli in transformed flies in addition to glomerulus DA4, which is the regular target of Or43a expressing neurons. Our results demonstrate the relevance of specific olfactory sensory input and subsequent processing in the antennal lobe for Drosophila behavior.     

Mating-type locus control of killer toxins from Kluyveromyces lactis and Pichia acaciae

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.     

Clarifying the Role of the Rostral dmPFC/dACC in Fear/Anxiety: Learning,Appraisal or Expression?

Recent studies have begun to carve out a specific role for the rostral part of the dorsal medial prefrontal cortex (dmPFC) and adjacent dorsal anterior cingulate cortex (dACC) in fear/anxiety. Within a novel general framework of dorsal mPFC/ACC areas subserving the appraisal of threat and concomitant expression of fear responses and ventral mPFC/ACC areas subserving fear regulation, the rostral dmPFC/dACC has been proposed to specifically mediate the conscious, negative appraisal of threat situations including, as an extreme variant, catastrophizing. An alternative explanation that has not been conclusively ruled out yet is that the area is involved in fear learning. We tested two different fear expression paradigms in separate fMRI studies (study 1: instructed fear, study 2: testing of Pavlovian conditioned fear) with independent groups of healthy adult subjects. In both paradigms the absence of reinforcement precluded conditioning. We demonstrate significant BOLD activation of an identical rostral dmPFC/dACC area. In the Pavlovian paradigm (study 2), the area only activated robustly once prior conditioning had finished. Thus, our data argue against a role of the area in fear learning. We further replicate a repeated observation of a dissociation between peripheral-physiological fear responding and rostral dmPFC/dACC activation, strongly suggesting the area does not directly generate fear responses but rather contributes to appraisal processes. Although we succeeded in preventing extinction of conditioned responding in either paradigm, the data do not allow us to definitively exclude an involvement of the area in fear extinction learning. We discuss the broader implications of this finding for our understanding of mPFC/ACC function in fear and in negative emotion more generally.     

Barley Rom1 antagonizes Rar1 function in Magnaporthe oryzae-infected leaves by enhancing epidermal and diminishing mesophyll defence

* Barley (Hordeum vulgare) is a host for Blumeria graminis f. sp. hordei (Bgh), which causes powdery mildew, and for the rice blast pathogen Magnaporthe oryzae. It has previously been shown that Rar1, initially identified in a mutational screen as being required for Mla12-specified Bgh-resistance, also controlled pathogenic growth of M. oryzae in barley. Here, we tested whether the rom1 mutation (restoration of Mla12-specified resistance), which restored resistance against Bgh in a susceptible rar1-2 genetic background, also influences the interaction between barley and M. oryzae. * Disease severity after infection with M. oryzae was analysed on rar1-2 mutants and rar1-2 rom1 double mutants. Microscopy and northern analysis were used to gain insight into cellular and molecular events. * On rar1-2 rom1 double mutant plants, the number of M. oryzae disease lesions was increased in comparison to the wild-type and the rar1-2 mutant which correlated with augmented epidermal penetration. However, a decrease in the lesion diameter, apparently conditioned in the mesophyll, was also observed. * These results highlight the impact of Rom1 in basal defence of barley against different pathogens. Importantly, a tissue-specific function for Rom1 with contrasting effects on epidermal and mesophyll defence was demonstrated.     

Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin.

We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.     

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.     

Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.