Direct Visualization of the Human Estrogen Receptor α Reveals a Role for Ligand in the Nuclear Distribution of the Receptor

The human estrogen receptor α (ER α) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER− (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER− but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the “unoccupied” and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus)

In the goldfish (Carassius auratus) the two endogenous forms of gonadotropin-releasing hormone (GnRH), namely chicken GnRH II ([His5,Trp7,Tyr8]GnRH) and salmon GnRH ([Trp7,Leu8]GnRH), stimulate the release of both gonadotropins and growth hormone from the pituitary. This control is thought to occur by means of the stimulation of distinct GnRH receptors. These receptors can be distinguished on the basis of differential gonadotropin and growth hormone releasing activities of naturally occurring GnRHs and GnRHs with variant amino acids in position 8. We have cloned the cDNAs of two GnRH receptors, GfA and GfB, from goldfish brain and pituitary. Although the receptors share 71% identity, there are marked differences in their ligand selectivity. Both receptors are expressed in the pituitary but are differentially expressed in the brain, ovary, and liver. Thus we have found and cloned two full-length cDNAs that appear to correspond to different forms of GnRH receptor, with distinct pharmacological characteristics and tissue distribution, in a single species.

Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides.

In tetrapods, only one gene encoding a somatostatin precursor has been identified so far. The present study reports the characterization of the cDNA clones that encode two distinct somatostatin precursors in the brain of the frog Rana ridibunda. The cDNAs were isolated by using degenerate oligonucleotides based on the sequence of the central region of somatostatin to screen a frog brain cDNA library. One of the cDNAs encodes a 115-amino acid protein (prepro-somatostatin-14; PSS1) that exhibits a high degree of structural similarity with the mammalian somatostatin precursor. The other cDNA encodes a 103-amino acid protein (prepro-[Pro2, Met13]somatostatin-14; PSS2) that contains the sequence of the somatostatin analog (peptide SS2) at its C terminus, but does not exhibit appreciable sequence similarity with PSS1 in the remaining region. In situ hybridization studies indicate differential expression of the PSS1 and PSS2 genes in the septum, the lateral part of the pallium, the amygdaloid complex, the posterior nuclei of the thalamus, the ventral hypothalamic nucleus, the torus semicircularis and the optic tectum. The somatostatin variant SS2 was significantly more potent (4-6 fold) than somatostatin itself in displacing [125I-Tyr0, D-Trp8] somatostatin-14 from its specific binding sites. The present study indicates that the two somatostatin variants could exert different functions in the frog brain and pituitary. These data also suggest that distinct genes encoding somatostatin variants may be expressed in the brain of other tetrapods.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Electrophoretic variation in adenylate kinase of Neisseria meningitidis is due to inter- and intraspecies recombination.

In prokaryotic and eukaryotic organisms, the electrophoretic variation in housekeeping enzymes from natural populations is assumed to have arisen by the accumulation of stochastic predominantly neutral mutations. In the naturally transformable bacterium Neisseria meningitidis, we show that variation in the electrophoretic mobility of adenylate kinase is due to inter- and intraspecies recombination rather than mutation. The nucleotide sequences of the adenylate kinase gene (adk) from isolates that express the predominant slow electrophoretic variant were rather uniform, differing in sequence at an average of 1.1% of nucleotide sites. The adk sequences of rare isolates expressing the fast migrating variant were identical to each other but had a striking mosaic structure when compared to the adk genes from strains expressing the predominant variant. Thus the sequence from the fast variants was identical to those of typical slow variants in the first 158 bp of the gene but differed by 8.4% in the rest of the gene (nt 159-636). The fast electrophoretic variant appears to have arisen by the replacement of most of the meningococcal gene with the corresponding region from the adk gene of a closely related Neisseria species. The adk genes expressing the electrophoretic variant with intermediate mobility were perfect, or almost perfect, recombinants between the adk genes expressing the fast and slow variants. Recombination may, therefore, play a major role in the generation of electrophoretically detectable variation in housekeeping enzymes of some bacterial species.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

Method for targetless tracking subpixel in-plane movements

We present a targetless motion tracking method for detecting planar movements with subpixel accuracy. This method is based on the computation and tracking of the intersection of two nonparallel straight-line segments in the image of a moving object in a scene. The method is simple and easy to implement because no complex structures have to be detected. It has been tested and validated using a lab experiment consisting of a vibrating object that was recorded with a high-speed camera working at 1000 fps. We managed to track displacements with an accuracy of hundredths of pixel or even of thousandths of pixel in the case of tracking harmonic vibrations. The method is widely applicable because it can be used for distance measuring amplitude and frequency of vibrations with a vision system.

New Orleans, Louisiana, Water Distribution System, 1902 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of the city of New Orleans : showing proposed water distribution system, [by] Sewerage and Water Board New Orleans, LA.; Geo. G. Earl, genl. sup't. It was published by the Sewerage and Water Board New Orleans in 1902. Scale [ca. 1:50,900]. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Louisiana State Plane Coordinate System, South NAD83 (in Feet) (Fipszone 1702). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows water distribution features such as existing and proposed water mains (with sizes), suction pipes, and water purification station sites. Also shows other features such as roads, canals, levees, drainage, cemeteries, Parish boundaries, and more. Shaded to show built-up and unbuilt areas for construction. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Particel size distribution and microstructures of sediments from ODP Leg 205 sites (Table 1)

Ocean Drilling Program Legs 170 and 205 offshore Costa Rica provide structural observations which support a new model for the geometry and deformation response to the seismic cycle of the frontal sedimentary prism and decollement. The model is based on drillcore, thin section, and electron microscope observations. The decollement damage zone is a few tens of meters in width, it develops mainly within the frontal prism. A clear cm-thick fault core is observed 1.6 km from the trench. The lower boundary of the fault core is coincident with the lithological boundary between the frontal prism and the hemipelagic and pelagic sediment of the Cocos plate. Breccia clast distributions in the upper portion of the decollement damage zone were studied through fractal analysis. This analysis shows that the fractal dimension changes with brecciated fragment size, implying that deformation was not accommodated by self-similar fracturing. A higher fractal dimensionality correlates with smaller particle size, which indicates that different or additional grain-size reduction processes operated during shearing. The co-existence of two distinct fracturing processes is also confirmed by microscopic analysis in which extension fracturing in the upper part of the damage zone farthest from the fault core is frequent, while both extension and shear fracturing occur approaching the fault core. The coexistence of extensional and shear fracturing seems to be best explained by fluid pressure variations in response to variations of the compressional regime during the seismic cycle. During the co-seismic event, sub-horizontal compression and fluid pressure increase, triggering shear fracturing and fluid expulsion. Fractures migrate upward with fluids, contributing to the asymmetric shape of the decollement, while slip propagates. In the inter-seismic interval the frontal prismrelaxes and fluid pressure drops. The frontal prismgoes into diffuse extension during the intervalwhen plate convergence is accommodated by creep along the ductile fault core. The fault core is typically a barrier to deformation, which is explained by its weak, but impermeable, nature. The localized development of a damage zone beneath the fault core is characterized by shear fracturing that appears as the result of local strengthening of the detachment.

Modelling the growth of prion protein aggregates in the brain in variant Creutzfeldt-Jakob disease

Deposition of insoluble prion protein (PrP) in the brain in the form of protein aggregates or deposits is characteristic of the ‘transmissible spongiform encephalopathies’ (TSEs). Understanding the growth and development of these PrP aggregates is important both in attempting to the elucidate of the pathogenesis of prion disease and in the development of treatments designed to prevent or inhibit the spread of prion pathology within the brain. Aggregation and disaggregation of proteins and the diffusion of substances into the developing aggregates (surface diffusion) are important factors in the development of protein aggregates. Mathematical models suggest that if aggregation/disaggregation or surface diffusion is the predominant factor, the size frequency distribution of the resulting protein aggregates in the brain should be described by either a power-law or a log-normal model respectively. This study tested this hypothesis for two different types of PrP deposit, viz., the diffuse and florid-type PrP deposits in patients with variant Creutzfeldt-Jakob disease (vCJD). The size distributions of the florid and diffuse plaques were fitted by a power-law function in 100% and 42% of brain areas studied respectively. By contrast, the size distributions of both types of plaque deviated significantly from a log-normal model in all brain areas. Hence, protein aggregation and disaggregation may be the predominant factor in the development of the florid plaques. A more complex combination of factors appears to be involved in the pathogenesis of the diffuse plaques. These results may be useful in the design of treatments to inhibit the development of protein aggregates in vCJD.

Size frequency distributions of the florid prion protein aggregates in variant Creutzfeldt-Jakob disease follow a power-law function

The objective was to test the hypothesis that the size frequency distributions of the prion protein (PrP) plaques in cases of variant Creutzfeldt-Jakob disease (vCJD) follow a power-law function. The design was a retrospective neuropathological study. The patients were 11 cases of clinically and neuropathologically verified vCJD. Size distributions of the diffuse and florid-type plaques were measured in several areas of the cerebral cortex and hippocampus from each case and a power-law function fitted to each distribution. The size distributions of the florid and diffuse plaques were fitted successfully by a powerlaw function in 100% and 42% of brain areas investigated respectively. Processes of aggregation/disaggregation may be more important than surface diffusion in the pathogenesis of the florid plaques. By contrast, surface diffusion may be a more significant factor in the development of the diffuse plaques. © Springer-Verlag Italia 2006.

Does the neuropathology of human patients with variant Creutzfeldt-Jakob disease reflect haematogenous spread of the disease?

To test the hypothesis that the distribution of the pathology in variant Creutzfeldt-Jakob disease (vCJD) represents haematogenous spread of the disease, we studied the spatial correlation between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles in the cerebral cortex, hippocampus, dentate gyrus, and cerebellum of 11 cases of the disease. In the majority of areas, there were no significant spatial correlations between either the vacuolation or the diffuse type of PrP deposit and the blood vessels. By contrast, a consistent pattern of spatial correlation was observed between the florid PrP deposits and blood vessels mainly in the cerebral cortex. The frequency of positive spatial correlations was similar in different anatomical areas of the cerebral cortex and in the upper compared with the lower laminae. Hence, with the exception of the florid deposits, the data do not demonstrate a spatial relationship between the pathological features of vCJD and blood vessels. The spatial correlation of the florid deposits and blood vessels may be attributable to factors associated with the blood vessels that promote the aggregation of PrP to form a condensed core rather than reflecting the haematogenous spread of the disease. © 2003 Elsevier Ireland Ltd. All rights reserved.

The spatial patterns of prion protein deposits in cases of variant Creutzfeldt-Jakob disease

The spatial patterns of the prion protein (PrP) deposits were studied in immunostained sections of areas of the cerebral cortex, hippocampus, dentate gyrus, and the molecular layer of the cerebellum in 11 cases of variant Creutzfeldt-Jakob disease (vCJD). Clustering of PrP deposits, with a regular distribution of the clusters parallel to the tissue boundary, was the most common spatial pattern observed. Two morphological types of PrP deposit were recognised, those consisting of a condensed core (florid deposits) and those deposits lacking a condensed core (non-florid deposits). The florid and non-florid PrP deposits exhibited a different profile of spatial patterns. First, the florid deposits exhibited a regularly distributed pattern of clusters more frequently than the non-florid deposits. Second, the florid deposits formed larger clusters (greater than1,600 µm in diameter) less frequently than the non-florid deposits. In the areas of the cerebral cortex that exhibited a regular distribution of PrP deposit clusters, the cluster size of the deposits approximated that of the groups of cells of the cortico-cortical pathway origin in only 12% of analyses. No significant differences in the frequency of the different types of spatial pattern were observed in different brain regions, or in the cerebral cortex between the upper and lower laminae. It was concluded that the spatial patterns of the PrP deposits in the cerebral cortex in vCJD are unlikely to reflect the degeneration of the cortico-cortical pathways as has been reported in sporadic CJD (sCJD). In addition, different factors could be involved in the development of the deposits with and without a condensed core.

The spatial pattern of the vacuolation ('spongiform change') in the cerebral cortex in variant Creutzfeldt-Jakob disease (vCJD)

The spatial pattern of the vacuolation ('spongiform change') was studied in areas of the cerebral cortex in 11 cases of variant Creutzfeldt-Jakob disease (vCJD). The vacuoles were evenly distributed along the cortex in 40/106 (38%) areas studied and randomly distributed in 6/106 (5.6%) areas. In 22/106 (21%) areas, the vacuoles were aggregated into clusters, 50 - 1600 μm in diameter and which were distributed in a regular pattern parallel to the pia mater. In 38/106 (36%) areas, large clusters of vacuoles, at least 1600 μm in diameter, were present. No significant differences in spatial patterns were observed between the different cortical regions or between the upper and lower laminae. In addition, age at onset and duration of the disease had no significant affect on spatial patterns. The spatial distribution of the vacuolation contrasts with that reported in sporadic CJD (sCJD) suggesting a different pattern of cortical degeneration in vCJD.

Quantitative variations in the pathology of 11 cases of variant Creutzfeldt-Jakob disease (vCJD)

Quantitative variations in the density and distribution of the vacuolation ('spongiform change'), surviving neurons, and prion protein (PrP) deposits were studied in eight brain regions from 11 cases of variant Creutzfeldt-Jakob disease (vCJD). Principal components analysis (PCA) was used to study the similarities and differences between cases and to identify the neuropathological variables which could best account for these variations. Two principal components (PC) were extracted from the data accounting in total for 93.4% of the variance; the majority of the variance (90%) being associated with PC1. Some clustering of the 11 cases in relation to PC1 and PC2 was evident. The densities of the vacuolation in the occipital cortex and the molecular layer of the cerebellum were positively and negatively correlated, respectively, with PC1. No significant variation between cases was associated with PrP deposition. These data suggest that vCJD cases have a consistent neuropathological profile characterised by the presence of vacuolation, neuronal loss and PrP deposition in the form of florid and non-florid deposits. However, there are quantitative variations between cases in the development of the vacuolation especially affecting the occipital cortex and cerebellum. © 2002 Elsevier Science Ireland Ltd. All rights reserved.