A genetic selection for circadian output pathway mutations in Neurospora crassa

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.     

APOBEC3G genetic variants and their influence on the progression to AIDS

The cytosine deaminase APOBEC3G, in the absence of the human immunodeficiency virus type 1 (HIV-1) accessory gene HIV-1 viral infectivity factor (vif), inhibits viral replication by introducing G-->A hypermutation in the newly synthesized HIV-1 DNA negative strand. We tested the hypothesis that genetic variants of APOBEC3G may modify HIV-1 transmission and disease progression. Single nucleotide polymorphisms were identified in the promoter region (three), introns (two), and exons (two). Genotypes were determined for 3,073 study participants enrolled in six HIV-AIDS prospective cohorts. One codon-changing variant, H186R in exon 4, was polymorphic in African Americans (AA) (f = 37%) and rare in European Americans (f < 3%) or Europeans (f = 5%). For AA, the variant allele 186R was strongly associated with decline in CD4 T cells (CD4 slope on square root scale: -1.86, P = 0.009), The 186R allele was also associated with accelerated progression to AIDS-defining conditions in AA. The in vitro antiviral activity of the 186R enzyme was not inferior to that of the common H186 variant. These studies suggest that there may be a modifying role of variants of APOBEC3G on HIV-1 disease progression that warrants further investigation.     

Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins

We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.     

Natural Occurrence of Seiridium cardinale on Thuja in Germany

Seiridium cardinale (Wagener) Sutton & Gibson (syn. Coryneum cardinale Wagener) is the most dangerous parasitic fungus of cypress and other Gupressaceae, already appearing in epidemic proportions, especially in the mediterranean area. Recently (in 1989, 1990 and 1991) this pathogen has been detected in the northern part of Germany (Hamburg and Schleswig-Holstein) naturally infecting Thuja. The geographical distribution and the progressive spread of S. cardinale is reviewed. Diagnostic features of the fungus, modes of host penetration, principal symptoms of the disease, ways of dispersal and control methods are described. Possible causal factors for the recently observed occurrence of 5. cardinale in the north of Germany are discussed.     

Non-consecutive versus consecutive footstrikes as an equivalent method of assessing gait asymmetry

Asymmetry of gait is often studied to characterize populations and assess the efficacy of treatment protocols. However, despite the continuous nature of gait, many studies have made comparisons between data from non-consecutive footstrikes. This is typically considered a limitation of these studies. However, if gait characteristics are sufficiently repeatable within a side, consecutive footstrikes may not be necessary to properly describe the asymmetry between sides. Therefore, one purpose of this study was to compare asymmetry values calculated from consecutive and non-consecutive footstrikes. Additionally, the variability of gait within and between sides was compared to assess the repeatability and distinctiveness of the characteristics on each side. The results suggest that kinetic and kinematic asymmetry can be assessed from either consecutive or non-consecutive footstrikes. Further, the patterns of movement tend to be sufficiently consistent within a side, such that the variability within a side is much lower than the variability between sides. However, there may be some variables, or populations, that exhibit high within-side variability. Several trials of consecutive footstrikes may be a better way to characterize asymmetry of those variables.     

The timing of differentiation of adult hippocampal neurons is crucial for spatial memory

Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3–4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.     

Human invariant NKT cells display alloreactivity instructed by invariant TCR-CD1d interaction and killer Ig receptors

Invariant NKT (iNKT) cells are a subset of highly conserved immunoregulatory T cells that modify a variety of immune responses, including alloreactivity. Central to their function is the interaction of the invariant TCR with glycosphingolipid (GSL) ligands presented by the nonpolymorphic MHC class I molecule CD1d and their ability to secrete rapidly large amounts of immunomodulatory cytokines when activated. Whether iNKT cells, like NK and conventional T cells, can directly display alloreactivity is not known. We show in this study that human iNKT cells and APC can establish a direct cross-talk leading to preferential maturation of allogeneic APC and a considerably higher reactivity of iNKT cells cultured with allogeneic rather that autologous APC. Although the allogeneic activation of iNKT cells is invariant TCR-CD1d interaction-dependent, GSL profiling suggests it does not involve the recognition of disparate CD1d/GSL complexes. Instead, we show that contrary to previous reports, iNKT cells, like NK and T cells, express killer Ig receptors at a frequency similar to that of conventional T cells and that iNKT cell allogeneic activation requires up-regulation and function of activating killer Ig receptors. Thus, iNKT cells can display alloreactivity, for which they use mechanisms characteristic of both NK and conventional T cells.     

A test on different aspects of diatom processing techniques

A factorial experiment was carried out in order to assess the effects of four treatment steps in diatom processing techniques (the quantity of hydrogen peroxide added to the sample, the time allowed to the digestion reaction, the use of centrifugation and the method used to settle the frustules onto the coverslip) on different aspects of the quality of the resulting microscopic slide: the concentration of organic and inorganic matter, the density, randomness and homogeneity of the frustule distribution in the slide, and the abundance of broken frustules and girdle views. The results show no significant effects of any of the experimental treatments on the proportion of broken frustules in the samples. No significant effects were related to the duration of the oxidation reaction in any of the aspects evaluated in the quality of the slides. A sample:hydrogen peroxide ratio of 1:1 for a 6-h oxidizing reaction can be used to optimize the protocol. Despite being more time consuming, the use of successive decantations instead of centrifuging is more appropriate to achieve better results. Slide quality can also be improved by leaving the coverslips to dry at room temperature in order to improve the distribution of frustules.     

11-(pyridinylphenyl)steroids--a new class of mixed-profile progesterone agonists/antagonists

Recently, a new class (often referred to as SPRMs: selective progesterone receptor modulators) of progesterone receptor ligands with mixed agonist/antagonist properties has been described. Such compounds are envisaged, for example, as treatment for endometriosis, uterine fibroids, and leiomyomas. Existing SPRMs include Asoprisnil 1 and Uliprisnil acetate 2. In our hands, however, these compounds proved to have a predominantly or exclusively antagonistic in vitro profile, which may make this type of compound less attractive, for example, as contraceptives. We therefore aimed at a class of mixed-profile compounds that would show a more evenly balanced agonist/antagonist profile. A novel class of 11beta-[4-(heteroaryl)phenyl]-substituted pregnanes was identified that displayed the desired balance. Contrary to known SPRMs, this novel class of MPP (mixed-profile progestagen) was found to have a truly mixed activity, including a sizeable agonist component.