Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus.

The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.

The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.

Bclx regulates the survival of double-positive thymocytes.

The bclx gene has been shown to regulate programmed cell death in vitro. We now show that Bclx expression increases dramatically when T cells differentiate from CD4- CD8- (double negative) thymocytes to CD4+ CD8+ [double positive (DP)] thymocytes. In contrast single-positive (SP) thymocytes express negligible amounts of Bclx protein. This expression pattern contrasts with that of Bcl2, which is present in double-negative thymocytes, down-regulated in DP thymocytes, and reinduced upon maturation to SP thymocytes. Elimination of Bclx by gene targeting dramatically shortens the survival of DP thymocytes but not the survival of SP thymocytes or peripheral SP T cells. These data suggest that the induction of Bclx during thymic maturation plays a critical role in regulating the length of time DP thymocytes survive in the absence of selection.

On the nature of the galactic early-B hypergiants

Aims. Despite their importance to a number of astrophysical fields, the lifecycles of very massive stars are still poorly defined. In order to address this shortcoming, we present a detailed quantitative study of the physical properties of four early-B hypergiants (BHGs) of spectral type B1-4 Ia+; Cyg OB2 #12, ζ1 Sco, HD 190603 and BP Cru. These are combined with an analysis of their long-term spectroscopic and photometric behaviour in order to determine their evolutionary status. Methods. Quantitative analysis of UV–radio photometric and spectroscopic datasets was undertaken with a non-LTE model atmosphere code in order to derive physical parameters for comparison with apparently closely related objects, such as B supergiants (BSGs) and luminous blue variables (LBVs), and theoretical evolutionary predictions. Results. The long-term photospheric and spectroscopic datasets compiled for the early-B HGs revealed that they are remarkably stable over long periods ( ≥ 40 yrs), with the possible exception of ζ1 Sco prior to the 20th century; in contrast to the typical excursions that characterise LBVs. Quantitative analysis of ζ1 Sco, HD 190603 and BP Cru yielded physical properties intermediate between BSGs and LBVs; we therefore suggest that BHGs are the immediate descendants and progenitors (respectively) of such stars, for initial masses in the range ~30−60 M⊙. Comparison of the properties of ζ1 Sco with the stellar population of its host cluster/association NGC 6231/Sco OB1 provides further support for such an evolutionary scenario. In contrast, while the wind properties of Cyg OB2 #12 are consistent with this hypothesis, the combination of extreme luminosity and spectroscopic mass (~110 M⊙) and comparatively low temperature means it cannot be accommodated in such a scheme. Likewise, despite its co-location with several LBVs above the Humphreys-Davidson (HD) limit, the lack of long term variability and its unevolved chemistry apparently excludes such an identification. Since such massive stars are not expected to evolve to such cool temperatures, instead traversing an O4-6Ia → O4-6Ia+ → WN7-9ha pathway, the properties of Cyg OB2 #12 are therefore difficult to understand under current evolutionary paradigms. Finally, we note that as with AG Car in its cool phase, despite exceeding the HD limit, the properties of Cyg OB2 #12 imply that it lies below the Eddington limit – thus we conclude that the HD limit does not define a region of the HR diagram inherently inimical to the presence of massive stars.

On the nature of candidate luminous blue variables in M 33

Context. Luminous blue variables (LBVs) are a class of highly unstable stars that have been proposed to play a critical role in massive stellar evolution as well as being the progenitors of some of the most luminous supernovae known. However the physical processes underlying their characteristic instabilities are currently unknown. Aims. In order to provide observational constraints on this behaviour we have initiated a pilot study of the population of (candidate) LBVs in the Local Group galaxy M 33. Methods. To accomplish this we have obtained new spectra of 18 examples within M 33. These provide a baseline of ≥ 4 yr with respect to previous observations, which is well suited to identifying LBV outbursts. We also employed existing multi-epoch optical and mid-IR surveys of M 33 to further constrain the variability of the sample and search for the presence of dusty ejecta. Results. Combining the datasets reveals that spectroscopic and photometric variability appears common, although in the majority of cases further observations will be needed to distinguish between an origin for this behavour in short lived stochastic wind structure and low level photospheric pulsations or coherent long term LBV excursions. Of the known LBVs we report a hitherto unidentified excursion of M 33 Var C between 2001-5, while the transition of the WNLh star B517 to a cooler B supergiant phase between 1993−2010 implies an LBV classification. Proof-of-concept quantitative model atmosphere analysis is provided for Romano’s star; the resultant stellar parameters being consistent with the finding that the LBV excursions of this star are accompanied by changes in bolometric luminosity. The combination of temperature and luminosity of two stars, the BHG [HS80] 110A and the cool hypergiant B324, appear to be in violation of the empirical Humphreys-Davidson limit. Mid-IR observations demonstrate that a number of candidates appear associated with hot circumstellar dust, although no objects as extreme as η Car are identified. The combined dataset suggests that the criteria employed to identify candidate LBVs results in a heterogeneous sample, also containing stars demonstrating the B[e] phenomenon. Of these, a subset of optically faint, low luminosity stars associated with hot dust are of particular interest since they appear similar to the likely progenitor of SN 2008S and the 2008 NGC 300 transient (albeit suffering less intrinsic extinction). Conclusions. The results of such a multiwavelength observational approach, employing multiplexing spectrographs and supplemented with quantitative model atmosphere analysis, appears to show considerable promise in both identifying and characterising the physical properties of LBVs as well as other short lived phases of massive stellar evolution.

Index of all reports issued by Bureaus of Labor Statistics in the United States, prior to March 1, 1902

prepared under the direction of Carroll D. Wright ; for the use of the United States Department of Labor.

Comparative wages, prices, and cost of living: (from the Sixteenth annual report of the Massachusetts Bureau of Statistics of Labor, for 1885)

Historical review of wages and prices. 1752-1860 -- Comparative wages, prices, and cost of living: Massachusetts and Great Britain. 1860-1883.

Puget Sound and Seattle, Washington, 1889 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Seattle Harbor : Puget Sound Washington territory, issued May 1870 C.P. Patterson, superintendant; verification J.E. Hilgard, assistant in charge of the office; triangulation by J. S. Lawson assistant in 1874 based upon the primary triangulation by George Davidson, assistant in 1855-6; topography and hydrography by J.S. Lawson, assistant in 1874 & 5; resurvey of city of Seattle and water front by assist. J.J. Gilbert in 1886; additions by asst. Pratt in 1889; verifications of hydrology by Lieut. Comdr. W. H. Brownson U.S.N. inspector of hydrography. It was published by United States Coast and Geodetic Survey in July 1889. Scale 1:20,000. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Washington State Plane North Coordinate System HARN NAD83 (in Feet) (Fipszone 4601). 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, index maps, legends, or other information associated with the principal map. This map shows coastal features such as lighthouses, rocks, channels, points, coves, islands, bottom soil types, flats, wharves, and more. Includes also selected land features such as roads, railroads, drainage, land cover, selected buildings, towns, and more. Relief shown by contours and spot heights; depths by soundings. Includes notes, tables, and list of authorities. 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.