The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.     

Spatial variability and temporal trends in water‐use efficiency of European forests

The increasing carbon dioxide (CO₂) concentration in the atmosphere in combination with climatic changes throughout the last century are likely to have had a profound effect on the physiology of trees: altering the carbon and water fluxes passing through the stomatal pores. However, the magnitude and spatial patterns of such changes in natural forests remain highly uncertain. Here, stable carbon isotope ratios from a network of 35 tree‐ring sites located across Europe are investigated to determine the intrinsic water‐use efficiency (iWUE), the ratio of photosynthesis to stomatal conductance from 1901 to 2000. The results were compared with simulations of a dynamic vegetation model (LPX‐Bern 1.0) that integrates numerous ecosystem and land–atmosphere exchange processes in a theoretical framework. The spatial pattern of tree‐ring derived iWUE of the investigated coniferous and deciduous species and the model results agreed significantly with a clear south‐to‐north gradient, as well as a general increase in iWUE over the 20th century. The magnitude of the iWUE increase was not spatially uniform, with the strongest increase observed and modelled for temperate forests in Central Europe, a region where summer soil‐water availability decreased over the last century. We were able to demonstrate that the combined effects of increasing CO₂ and climate change leading to soil drying have resulted in an accelerated increase in iWUE. These findings will help to reduce uncertainties in the land surface schemes of global climate models, where vegetation–climate feedbacks are currently still poorly constrained by observational data.     

Characterization of the ATP-binding cassette transporter gene expression profile in Y79: a retinoblastoma cell line

The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH(2)-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a "closed" conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH(2)-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a "closed" conformational state of TES may be involved in nucleolar localization.     

On the use of an automated learning device by group-housed dwarf goats: Do goats seek cognitive challenges?

It has been postulated that implementing appropriate cognitive challenges in the housing of captive animals allows them to use their cognitive skills, and to learn to control certain aspects of their environment. This cognitive activity is thought to be a major source of positive emotions and a prerequisite for psychological well-being of the animal. We studied the use of an automated learning device (LD) by group-housed dwarf goats. To determine if the animals seek cognitive challenges, we investigated whether the goats continued to operate the LD to get a reward when the same amount of reward was available concurrently without additional cognitive effort. In a first training period (W), a group of 10 dwarf goats were trained to press a button at a waterer (WA) to get 35 ml of drinking water. In two following periods (P1 and P2), the goats learned to discriminate two different sets of visual shapes at the LD to get the same amount of water as a reward. The WA was closed in these periods. Finally, in two experimental periods (P2r and P3), water was concurrently available at the WA and the LD for five days. We analysed the use of the LD when the WA was accessible: (a) while the goats were presented with the discrimination problem that they had learned previously (P2r); and (b) while they were given a new problem to learn (P3). The total number of button presses that triggers the delivery of water (presses at the WA and/or successful presses at the LD) was stable throughout the study, whether water was delivered only at the waterer (WA) or, after successful shape discrimination, at the LD, or if it was concurrently available at both devices. When water was concurrently available at the WA and the LD (P2r, P3), the goats constantly directed 1/3 of all daily button presses to the LD. By doing so, they gained 23.0% (P2r) and 13.8% (P3) of daily drinking water at the LD because of differing learning success. Goats showed large individual differences in the number of button presses at the LD in P2r and P3. We found a strong correlation between individual learning success and the use of the LD in P2r (r_s = 0.75; P = 0.025) and P3 (r_s = 0.76; P = 0.036). Results indicate that cognitive challenges, and especially successful coping with the challenge, could have at least moderate intrinsic reinforcing properties beyond the reward.     

Effects of starvation on reproduction of the predacious mite <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae)

Effects of starvation on gravid females of Neoseiulus californicus were investigated at 20°C and 85% RH. When females that had been reared with abundant prey were swapped, just after laying their first egg, to conditions without any prey and water, they laid 1.8 eggs and survived for 4.3 days. In the body of well-fed females, an egg with eggshell and/or two oocytes were observed in the ventral and dorsal regions, respectively. The larger oocyte had two roundish nuclei and abundant yolk granules, and was enveloped with a vitelline membrane. These two nuclei were not fused but were just close to each other. The smaller oocyte had a nucleus, but had not yet formed yolk granules and vitelline membrane. Females after 12 h starvation had an egg in the ventral region and an oocyte in the dorsal region of the body. After more than 24 h starvation females maintained an oocyte in the dorsal region of the body, but had no egg in the ventral region. The oocyte was filled with abundant yolk granules and contained two irregular nuclei when females were starved for 24 h, but when starved for more than 36 h it contained one irregular nucleus. These findings suggest that (1) gravid females maintained an oocyte in the dorsal region after laying two eggs during starvation, (2) the oocyte was not absorbed during starvation, (3) the oocyte advanced vitellogenesis and the fusion of two nuclei, and (4) the vitellogenic oocyte was not enveloped with an eggshell and had not started embryogenesis.     

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.     

Formation of palladium(0) nanoparticles at microbial surfaces

The increasing demand and limited natural resources for industrially important platinum‐group metal (PGM) catalysts render the recovery from secondary sources such as industrial waste economically interesting. In the process of palladium (Pd) recovery, microorganisms have revealed a strong potential. Hitherto, bacteria with the property of dissimilatory metal reduction have been in focus, although the biochemical reactions linking enzymatic Pd(II) reduction and Pd(0) deposition have not yet been identified. In this study we investigated Pd(II) reduction with formate as the electron donor in the presence of Gram‐negative bacteria with no documented capacity for reducing metals for energy production: Cupriavidus necator, Pseudomonas putida, and Paracoccus denitrificans. Only large and close‐packed Pd(0) aggregates were formed in cell‐free buffer solutions. Pd(II) reduction in the presence of bacteria resulted in smaller, well‐suspended Pd(0) particles that were associated with the cells (called “bioPd(0)” in the following). Nanosize Pd(0) particles (3–30 nm) were only observed in the presence of bacteria, and particles in this size range were located in the periplasmic space. Pd(0) nanoparticles were still deposited on autoclaved cells of C. necator that had no hydrogenase activity, suggesting a hydrogenase‐independent formation mechanism. The catalytic properties of Pd(0) and bioPd(0) were determined by the amount of hydrogen released in a reaction with hypophosphite. Generally, bioPd(0) demonstrated a lower level of activity than the Pd(0) control, possibly due to the inaccessibility of the Pd(0) fraction embedded in the cell envelope. Our results demonstrate the suitability of bacterial cells for the recovery of Pd(0), and formation and immobilization of Pd(0) nanoparticles inside the cell envelope. However, procedures to make periplasmic Pd(0) catalytically accessible need to be developed for future nanobiotechnological applications. Biotechnol. Bioeng. 2010;107: 206–215. © 2010 Wiley Periodicals, Inc.     

Murine Features of Neurogenesis in the Human Hippocampus across the Lifespan from 0 to 100 Years

Background

Essentially all knowledge about adult hippocampal neurogenesis in humans still comes from one seminal study by Eriksson et al. in 1998, although several others have provided suggestive findings. But only little information has been available in how far the situation in animal models would reflect the conditions in the adult and aging human brain. We therefore here mapped numerous features associated with adult neurogenesis in rodents in samples from human hippocampus across the entire lifespan. Such data would not offer proof of adult neurogenesis in humans, because it is based on the assumption that humans and rodents share marker expression patterns in adult neurogenesis. Nevertheless, together the data provide valuable information at least about the presence of markers, for which a link to adult neurogenesis might more reasonably be assumed than for others, in the adult human brain and their change with increasing age.

Methods and Findings

In rodents, doublecortin (DCX) is transiently expressed during adult neurogenesis and within the neurogenic niche of the dentate gyrus can serve as a valuable marker. We validated DCX as marker of granule cell development in fetal human tissue and used DCX expression as seed to examine the dentate gyrus for additional neurogenesis-associated features across the lifespan. We studied 54 individuals and detected DCX expression between birth and 100 years of age. Caveats for post-mortem analyses of human tissues apply but all samples were free of signs of ischemia and activated caspase-3. Fourteen markers related to adult hippocampal neurogenesis in rodents were assessed in DCX-positive cells. Total numbers of DCX expressing cells declined exponentially with increasing age, and co-expression of DCX with the other markers decreased. This argued against a non-specific re-appearance of immature markers in specimen from old brains. Early postnatally all 14 markers were co-expressed in DCX-positive cells. Until 30 to 40 years of age, for example, an overlap of DCX with Ki67, Mcm2, Sox2, Nestin, Prox1, PSA-NCAM, Calretinin, NeuN, and others was detected, and some key markers (Nestin, Sox2, Prox1) remained co-expressed into oldest age.

Conclusions

Our data suggest that in the adult human hippocampus neurogenesis-associated features that have been identified in rodents show patterns, as well as qualitative and quantitative age-related changes, that are similar to the course of adult hippocampal neurogenesis in rodents. Consequently, although further validation as well as the application of independent methodology (e.g. electron microscopy and cell culture work) is desirable, our data will help to devise the framework for specific research on cellular plasticity in the aging human hippocampus.     

HIF-1beta determines ABCA1 expression under hypoxia in human macrophages

ATP-binding cassette transporter A1 plays (ABCA1) a major role in reverse cholesterol transport, a process closely related to atherogenesis. In the thickening atherosclerotic lesions lipid loaded macrophages are exposed to regions of local hypoxia that may influence reverse cholesterol transport. Here we studied the effect of hypoxia on ABCA1 regulation and cholesterol efflux in human macrophages.We found that the hypoxia-inducible factor 1 (HIF-1) specifically binds to the HIF-1 response element of the ABCA1 promoter and the HIF-1 complex increases ABCA1 promoter activity along with ABCA1 expression. Primary human macrophages exposed to hypoxia or expressing constitutively active HIF-1alpha responded with a potent change in ABCA1 expression, which showed a strong correlation with HIF-1beta expression (r: 0.95–0.91). Moreover, ABCA1-mediated cholesterol efflux was also found to be regulated by HIF-1beta under hypoxia. In vivo, in macrophages prepared from human atherosclerotic lesions ABCA1 levels showed a strong correlation with HIF-1beta expression. This in vivo regulatory mechanism was confirmed in human pre-eclamptic placentas, a clinical condition with severe local hypoxia.These results demonstrate that HIF-1beta availability determines ABCA1 expression and cholesterol efflux in macrophages under hypoxia and may contribute to the interpersonal variability of atherosclerotic lesion progression.