Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays,high resolution mass spectrometry and off-line NMR

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatography–high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-¹H, 2D-COSY, 2D-NOESY, and ¹H-¹³C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.     

EstB-mediated hydrolysis of the siderophore triacetylfusarinine C optimizes iron uptake of Aspergillus fumigatus

Aspergillus fumigatus excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three N(5)-cis-anhydromevalonyl-N(5)-hydroxy-N(2)-acetyl-l-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene, estB, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of estB in A. fumigatus eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo. Escherichia coli-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three N(2)-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in A. fumigatus.     

Induction of Empathy by the Smell of Anxiety

The communication of stress/anxiety between conspecifics through chemosensory signals has been documented in many vertebrates and invertebrates. Here, we investigate how chemosensory anxiety signals conveyed by the sweat of humans (N = 49) awaiting an academic examination are processed by the human brain, as compared to chemosensory control signals obtained from the same sweat donors in a sport condition. The chemosensory stimuli were pooled according to the donation condition and administered to 28 participants (14 males) synchronously to breathing via an olfactometer. The stimuli were perceived with a low intensity and accordingly only about half of the odor presentations were detected by the participants. The fMRI results (event-related design) show that chemosensory anxiety signals activate brain areas involved in the processing of social emotional stimuli (fusiform gyrus), and in the regulation of empathic feelings (insula, precuneus, cingulate cortex). In addition, neuronal activity within attentional (thalamus, dorsomedial prefrontal cortex) and emotional (cerebellum, vermis) control systems were observed. The chemosensory perception of human anxiety seems to automatically recruit empathy-related resources. Even though the participants could not attentively differentiate the chemosensory stimuli, emotional contagion seems to be effectively mediated by the olfactory system.     

Nitrosomonas europaea expresses a nitric oxide reductase during nitrification

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.     

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C,extracellular signal-regulated kinase,and phospholipase A2

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.     

The glucose transport facilitator GLUT8 is predominantly associated with the acrosomal region of mature spermatozoa

The glucose transporter 8 (GLUT8) is a recently identified member of the family of sugar transport facilitators. In human tissues GLUT8 is predominantly expressed in testis in a gonadotropin-dependent manner. It is shown here that the onset of mRNA synthesis of GLUT8 during the maturation of mouse testis coincides with the appearance of mature spermatozoa. Furthermore, immunohistochemistry with antiserum against the C-terminus of GLUT8 indicated that the protein was associated with spermatozoa within the seminiferous and the epididymal tubules. The GLUT8 immunoreactivity was detected within the head of mouse and human spermatozoa in the acrosomal region, and appeared to be located at the plasma membrane as well as within the cells. This specific expression and localization of GLUT8 suggests that the transport facilitator plays a major role in the fuel supply of mature spermatozoa, and that it is a potential target for inhibition of sperm cell function.