bluuebunny/crossref_metadata_2025 huggingface dataset:
Shifts in Strategic Spatial Planning? Some Evidence from Europe and Australia
Authors: Albrechts Louis
Published: June 2006

Clear-cut definitions of strategic spatial planning are rather exceptional. Therefore in this paper I use building blocks from literature (planning and business) and my experience in practice to construct a workable normative definition of the ‘what’ and ‘how’ of strategic spatial planning. Five main characteristics (selective, relational annex inclusive, integrative, visioning, and action orientated) that constitute the hard core of the ‘strategic’ in the normative view are confronted, in a first broad scan, with nine so-called strategic plans from different planning traditions in Europe (Italy, France, Spain, the Czech Republic, Belgium, and the Netherlands) and in Australia (Perth). The confrontation highlights some hesitant shifts towards the normative view but also makes it clear that there is still a long way to go.
Job-Housing Mismatch: Affordability Crisis in Surrey, South East England
Authors: Morrison Nicola , Monk Sarah
Published: June 2006

Rising housing costs and lack of affordable housing in London are affecting labour recruitment and retention, particularly in the public sector—the ‘key-worker problem’ of recent government statements. But housing and labour-market problems are not confined to London. In this paper we highlight the extent and nature of affordability, recruitment, and retention problems in a high-cost location outside the capital: Surrey in South East England. We suggest that changes towards a more professional and managerial workforce have impacted on housing demand and hence prices. Planning and physical constraints have contributed to housing shortages and exacerbated rising housing costs. High housing costs are linked to recruitment and retention difficulties not only for public-sector employers but also for certain types of private-sector employers in Surrey. This raises problems for the sustainability of the local economy. Measures introduced to address these difficulties are helping ‘key workers’ to access housing locally, but this simply bids up house prices rather than increasing the overall supply of housing that is affordable.
Clusters, surfaces, and catalysis
Authors: Somorjai Gabor A. , Contreras Anthony M. , Montano Max , Rioux Robert M.
Published: June 2006

The surface science of heterogeneous metal catalysis uses model systems ranging from single crystals to monodispersed nanoparticles in the 1–10 nm range. Molecular studies reveal that bond activation (C–H, H–H, C–C, C

O) occurs at 300 K or below as the active metal sites simultaneously restructure. The strongly adsorbed molecules must be mobile to free up these sites for continued turnover of reaction. Oxide–metal interfaces are also active for catalytic turnover. Examples using C–H and C

O activation are described to demonstrate these properties. Future directions include synthesis, characterization, and reaction studies with 2D and 3D monodispersed metal nanoclusters to obtain 100% selectivity in multipath reactions. Investigations of the unique structural, dynamic, and electronic properties of nanoparticles are likely to have major impact in surface technologies. The fields of heterogeneous, enzyme, and homogeneous catalysis are likely to merge for the benefit of all three.
Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic
Authors: Raqib Rubhana , Sarker Protim , Bergman Peter , Ara Gul , Lindh Monica , Sack David A. , Nasirul Islam K. M. , Gudmundsson Gudmundur H. , Andersson Jan , Agerberth Birgitta
Published: June 2006

Shigella
is a major cause of morbidity, mortality, and growth retardation for children in developing countries. Emergence of antibiotic resistance among
Shigellae
demands the development of effective medicines. Previous studies found that the endogenous antimicrobial peptide LL-37 is down-regulated in the rectal epithelium of patients during shigellosis and that butyrate up-regulates the expression of LL-37 in colonic epithelial cells
in vitro
and decreases severity of inflammation in experimental shigellosis. In this study,
Shigella
-infected dysenteric rabbits were treated with butyrate (0.14 mmol/kg of body weight) twice daily for 3 days, and the expression levels of the rabbit homologue to LL-37, CAP-18, were monitored in the colon. Butyrate treatment resulted in (
i
) reduced clinical illness, severity of inflammation in the colon, and bacterial load in the stool, (
ii
) significant up-regulation of CAP-18 in the surface epithelium, and (
iii
) disappearance of CAP-18-positive cells in lamina propria. The active CAP-18 peptide was released in stool from its proform by butyrate treatment. In healthy controls, CAP-18 expression was localized predominantly to the epithelial surface of the colon. In infected rabbits, CAP-18 expression was localized to immune and inflammatory cells in the colon, whereas the ulcerated epithelium was devoid of CAP-18 expression. The combination of CAP-18 and butyrate was more efficient in killing
Shigella in vitro
than CAP-18 alone. Our findings indicate that oral butyrate treatment in shigellosis may be of clinical value because of induction of the endogenous cathelicidin CAP-18 in the colonic epithelium, stimulation of the release of the active peptide CAP-18, and promoting elimination of
Shigella
.
Solar modulation of Little Ice Age climate in the tropical Andes
Authors: Polissar P. J. , Abbott M. B. , Wolfe A. P. , Bezada M. , Rull V. , Bradley R. S.
Published: June 2006

The underlying causes of late-Holocene climate variability in the tropics are incompletely understood. Here we report a 1,500-year reconstruction of climate history and glaciation in the Venezuelan Andes using lake sediments. Four glacial advances occurred between
anno Domini
(A.D.) 1250 and 1810, coincident with solar-activity minima. Temperature declines of −3.2 ± 1.4°C and precipitation increases of ≈20% are required to produce the observed glacial responses. These results highlight the sensitivity of high-altitude tropical regions to relatively small changes in radiative forcing, implying even greater probable responses to future anthropogenic forcing.
Identifying genes important for spermatogonial stem cell self-renewal and survival
Authors: Oatley Jon M. , Avarbock Mary R. , Telaranta Aino I. , Fearon Douglas T. , Brinster Ralph L.
Published: June 2006

Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and, thus, preservation of a species. Because of stem cell rarity, studying their self-renewal is greatly facilitated by
in vitro
culture of enriched biologically active cell populations. A recently developed culture method identified glial cell line-derived neurotrophic factor (GDNF) as the essential growth factor that supports
in vitro
self-renewal of SSCs and results in an increase in their number. This system is a good model to study mechanisms of stem cell self-renewal because of the well defined culture conditions, enriched cell population, and available transplantation assay. By withdrawing and replacing GDNF in culture medium, we identified regulated expression of many genes by using microarray analysis. The expression levels of six of these genes were dramatically decreased by GDNF withdrawal and increased by GDNF replacement. To demonstrate the biological significance of the identified GDNF-regulated genes, we examined the importance of the most responsive of the six,
bcl6b
, a transcriptional repressor. By using siRNA to reduce transcript levels, Bcl6b was shown to be crucial for SSC maintenance
in vitro
. Moreover, evaluation of Bcl6b-null male testes revealed degeneration and/or absence of active spermatogenesis in 24 ± 7% of seminiferous tubules. These data suggest that Bcl6b is an important molecule in SSC self-renewal and validate the biological relevance of the GDNF-regulated genes identified through microarray analysis. In addition, comparison of data generated in this study to other stem cell types suggests that self-renewal in SSCs is regulated by distinctly different molecular mechanisms.
Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains.
Authors: Nishibe S , Wahl M I , Wedegaertner P B , Kim J W , Rhee S G , Carpenter G , Kim J J
Published: June 2006

Phosphatidylinositol-specific phospholipase C isozyme gamma (PLC-gamma, Mr 145,000) is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. PLC-beta-1, another PLC isozyme, is a poor substrate for the EGF receptor. We examined the relative phosphorylation of PLC-gamma and PLC-beta-1 by the 170-kDa native EGF receptor molecule, the 66-kDa cytoplasmic kinase domain of the EGF receptor (Arg647-Ala1186), the alpha 2 beta 2 native insulin receptor, and the 48-kDa cytoplasmic kinase domain of the insulin receptor beta subunit (Gly947-Ser1343). Similar to the intact EGF receptor, the cytoplasmic kinase domain of the EGF receptor preferentially phosphorylated PLC-gamma. High-performance liquid chromatographic comparison of tryptic phosphopeptides from PLC-gamma phosphorylated by both forms of the EGF receptor kinase indicated similar patterns of multiple tyrosine phosphorylations. These results imply that substrate selectivity, at least in terms of PLC isozymes, is independent of the extracellular ligand-binding and membrane anchor domains of the EGF receptor. In comparison, neither the intact insulin receptor nor the beta-chain kinase domain was able to phosphorylate PLC-gamma to a significant extent. Also, insulin failed to stimulate the phosphorylation of PLC-gamma in NIH 3T3/HIR cells, which overexpress the human insulin receptor. Thus PLC-gamma is not a phosphorylation substrate for the insulin receptor in vitro or in the intact cell.
Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.
Authors: Wiktor-Jedrzejczak W , Bartocci A , Ferrante A W , Ahmed-Ansari A , Sell K W , Pollard J W , Stanley E R
Published: June 2006

Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse.
Luteinizing hormone-releasing hormone neurons express Fos protein during the proestrous surge of luteinizing hormone.
Authors: Lee W S , Smith M S , Hoffman G E
Published: June 2006

The ability of luteinizing hormone-releasing hormone (LHRH) neurons to express the oncogene c-fos was examined during the estrous cycle in rats. The immunocytochemical localization of the c-fos-encoded antigen, Fos, was coupled with the immunocytochemical localization of LHRH. LHRH neurons showed no Fos immunoreactivity during diestrus-1, diestrus-2, estrus, or the morning of proestrus. However, Fos was expressed in LHRH neurons from 1600 to 2200 hours during proestrus. The timing of onset of Fos expression in LHRH neurons during proestrus suggests a strong correlation with increased LH secretion. Pentobarbital, which blocks the preovulatory LH surge, blocked Fos expression in LHRH neurons, but the LHRH neurons expressed Fos on the following afternoon at the time of the expected delayed LH surge. Not all LHRH neurons expressed Fos during the LHRH surge. Approximately half of the LHRH neurons were activated in the preoptic area and anterior hypothalamus; more anteriorly positioned LHRH neurons did not express Fos, resulting in an overall stimulation of 40% of the LHRH neurons. These data provide direct evidence that stimulation of LHRH neurons during proestrus takes place at the LHRH cell bodies, and identify the specific population of LHRH neurons which are activated.
Escherichia coli RecQ protein is a DNA helicase.
Authors: Umezu K , Nakayama K , Nakayama H
Published: June 2006

The Escherichia coli recQ gene, a member of the RecF recombination gene family, was set in an overexpression plasmid, and its product was purified to near-homogeneity. The purified RecQ protein exhibited a DNA-dependent ATPase and a helicase activity. Without DNA, no ATPase activity was detected. The capacity as ATPase cofactor varied with the type of DNA in the following order: circular single strand greater than linear single strand much greater than circular or linear duplex. As a helicase, RecQ protein displaced an annealed 71-base or 143-base single-stranded fragment from circular or linear phage M13 DNA, and the direction of unwinding seemed to be 3'----5' with respect to the DNA single strand to which the enzyme supposedly bound. Furthermore, the protein could unwind 143-base-pair blunt-ended duplex DNA at a higher enzyme concentration. It is concluded that RecQ protein is a previously unreported helicase, which might possibly serve to generate single-stranded tails for a strand transfer reaction in the process of recombination.
Role of early genes in pathogenesis of adenovirus pneumonia.
Authors: Ginsberg H S , Horswood R L , Chanock R M , Prince G A
Published: June 2006

Intranasal inoculation of type 5 adenovirus into the cotton rat Sigmodon hispidus produces a pneumonia pathologically similar to that in humans, and it, therefore, provides an excellent animal model to investigate the pathogenesis of this disease. The goal of this study was to test the hypothesis that accumulation of viral structural proteins is responsible for a major portion of the cell-damage-producing disease. Since viral DNA replication is essential for synthesis of the viral structural proteins, which are products of late genes, the hypothesis was tested using mutants defective in genes required for DNA synthesis. Most experiments were done with the conditionally lethal temperature-sensitive (ts) mutant H5ts125, which contains a mutation in the early region 2A (E2A) gene encoding the DNA-binding protein. The data show that infection with 1 x 10(9.0) plaque-forming units of H5ts125 induced a pneumonia that was as extensive and qualitatively the same as that after wild-type adenovirus type 5 infection, although H5ts125 did not replicate to produce infectious virus. When cotton rats were infected with 1 x 10(8.0) plaque-forming units of wild-type adenovirus type 5 or H5ts125, the pneumonias that followed were pathologically similar; in the latter phases, however, wild-type virus produced slightly more extensive pneumonia than did H5ts125, probably because its replication permitted infection of more susceptible cells.
Nonrandom duplication of the chromosome bearing a mutated Ha-ras-1 allele in mouse skin tumors.
Authors: Bianchi A B , Aldaz C M , Conti C J
Published: June 2006

We analyzed the normal/mutated allelic ratio of the Ha-ras-1 gene in mouse skin squamous cell carcinomas induced by initiation with dimethylbenz[a]anthracene and promotion with phorbol 12-myristate 13-acetate. DNA for these studies was obtained from short-term tumor cultures (24-72 hr) to eliminate the contribution of stromal and inflammatory cells to the sample. The allelotypic analysis was performed in 25 squamous cell carcinomas by quantitative radio-analysis of the Xba I restriction fragment length polymorphism as detected by BS9, a v-Ha-ras probe, and rehybridization of the Southern blots with probes for chromosomes 7 and 8. Approximately 85% of the tumors presented overrepresentation of the mutated allele in the form of 1 normal/2 mutated (12 tumors), 0 normal/3 mutated (4 tumors), 0 normal/2 mutated (3 tumors), and gene amplification (3 tumors). No tumor was found with a 2 normal/1 mutated allelic ratio. These results support our previous cytogenetic studies, indicating that trisomy of chromosome 7 is present in the majority of these tumors and show that nonrandom duplication of the chromosome carrying the mutated Ha-ras-1 allele appears to be a major mechanism by which the mutated gene is overrepresented.
Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle.
Authors: Bunnell B A , Heath L S , Adams D E , Lahti J M , Kidd V J
Published: June 2006

We have isolated and characterized cDNA encoding a human 58-kDa protein kinase that is homologous to the cell division control (CDC) protein kinases. This protein kinase also contains a unique N-terminal domain that may potentially regulate its function. Due to its relatedness to p34CDC2, the human p58 cDNA was overexpressed in CHO cells to determine the effect on the cell cycle. Elevated expression of p58 in these cells resulted in prolonged late telophase and early G1 phase of the cell cycle. These p58 overexpressors showed a significantly increased frequency of tubulin midbodies as well as significant increases in mitotic abnormalities. Thus, proper regulation of p58 protein kinase is essential for normal cell cycle progression in these cells.
Rare maternal mRNAs code for regulatory proteins that control lineage-specific gene expression in the sea urchin embryo.
Authors: Cutting A E , Höög C , Calzone F J , Britten R J , Davidson E H
Published: June 2006

The prevalence of mRNAs coding for the sea urchin embryo regulatory factors P3A1 and P3A2 was measured by single-strand probe excess solution hybridization. P3A1 and P3A2 are not homologous proteins, though they both bind specifically to a particular cis-regulatory sequence. Interaction at this target site is known to be required for lineage-specific expression of an aboral ectoderm-specific gene and probably for several other genes as well. Genome blot hybridizations show that both factors are encoded by single-copy genes. Maternal mRNAs for both factors are present at less than 10(3) molecules per egg, which places them in the rare mRNA class. During development to the mesenchyme blastula stage, the amount of P3A1 mRNA (per embryo) increases severalfold while that of P3A2 remains approximately constant. Specification of the aboral ectoderm founder cells and of their initial patterns of gene expression must occur during early to mid-cleavage stage. Therefore, the regulatory proteins needed for this process must be produced by this stage. We show that the quantities of the P3A proteins that can be synthesized from the numbers of mRNA molecules present in the large blastomeres of the early embryo are sufficient to be functional, because these proteins will be accumulated in the nuclei. Thus maternal P3A1 or P3A2 proteins asre not required, nor were these detected in earlier studies. Furthermore, differential spatial (as well as temporal) distribution of both of these newly synthesized factor species could result from the unequal cleavage pattern utilized in the sea urchin egg.
Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.
Authors: Rubin A L , Arnstein P , Rubin H
Published: June 2006

NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration.
Susceptibility or resistance to lysis by alloreactive natural killer cells is governed by a gene in the human major histocompatibility complex between BF and HLA-B.
Authors: Ciccone E , Colonna M , Viale O , Pende D , Di Donato C , Reinharz D , Amoroso A , Jeannet M , Guardiola J , Moretta A
Published: June 2006

The specificity recognized on normal allogeneic cells by a given alloreactive (1-anti-A) natural killer clone is controlled by a gene locus termed EC1. Because the EC1 locus was previously shown to be located on chromosome 6, families characterized by a recombinant major histocompatibility complex haplotype were analyzed to map this locus more precisely. The breakpoint of recombination was studied by standard HLA typing, complement typing, and restriction fragment length polymorphism analysis of a series of genes located between the complement cluster genes and HLA-B within the major histocompatibility complex region. Three of 10 families analyzed were informative. From the data obtained, the EC1 locus maps between BF and HLA-B and presumably is one of the normal genes recently described in this region.
Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly.
Authors: Abate C , Luk D , Gentz R , Rauscher F J , Curran T
Published: June 2006

The protein products of the fos and jun protooncogenes interact cooperatively in the form of a heterodimer with the activator protein 1 (AP-1) regulatory element. To characterize the properties of these proteins, we have expressed polypeptides comprised of the dimerization and DNA-binding domains of Fos and Jun in Escherichia coli. The mini-Fos (wbFos) and the mini-Jun (wbJun) proteins were purified to apparent homogeneity by using a nickel affinity chromatography procedure. Purified wbFos and wbJun associated rapidly in vitro and interacted cooperatively with the human metallothionein IIA AP-1-binding site. However, efficient DNA binding of wbJun and wbFos-wbJun complexes required an additional activity present in nuclear extracts. This activity was sensitive to alkylating agents and could be partially mimicked by the presence of reducing and stabilizing agents. DNase I footprinting experiments demonstrated that Jun homodimeric complexes and Fos-Jun heterodimeric complexes interacted with the same site on the human metallothionein IIA gene. Moreover, UV-crosslinking studies demonstrated that Fos and Jun contact DNA directly and that both proteins interacted equivalently with either strand of the AP-1-binding site.
A clonal theory of parasitic protozoa: the population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences.
Authors: Tibayrenc M , Kjellberg F , Ayala F J
Published: June 2006

We propose a general theory of clonal reproduction for parasitic protozoa, which has important medical and biological consequences. Many parasitic protozoa have been assumed to reproduce sexually, because of diploidy and occasional sexuality in the laboratory. However, a population genetic analysis of extensive data on biochemical polymorphisms indicates that the two fundamental consequences of sexual reproduction (i.e., segregation and recombination) are apparently rare or absent in natural populations of the parasitic protozoa. Moreover, the clones recorded appear to be stable over large geographical areas and long periods of time. A clonal population structure demands that the medical attributes of clones be separately characterized; ubiquitous clones call for priority characterization. Uniparental reproduction renders unsatisfactory Linnean taxonomy; this needs to be supplemented by the "natural clone" as an additional taxonomic unit, which is best defined by means of genetic markers.
A murine macrophage line of the H-2d/f haplotype can activate H-2k suppressor T cells.
Authors: Chang M D , Jaureguiberry B , Garrido E , Diamond B
Published: June 2006

Little is known about the molecular basis for activation of suppressor T cells. In this report we describe two macrophage cell lines, BAC1.2.SC8 and its variant progeny B26, that differ in their ability to activate suppressor T cells. The SC8 line is derived from a (BALB/c x A.CA)F1 (H-2d/f) mouse and is haploid with respect to I-Ed. It is capable of activating I-Ed-restricted helper T cells as well as poly-(Glu50Tyr50)-specific I-Ed-restricted suppressor cells. The B26 variant can activate H-2d-restricted helper T cells but activates H-2k-restricted suppressor cells. The I-Ed molecules of SC8 and of B26 have identical amino acid sequences. This suggests that suppressor T cells either recognize posttranslational modifications of the I-E molecule or that there is another accessory molecule that helps determine the major histocompatibility complex restriction in the activation of suppressor T cells.
Exon skipping during splicing of albumin mRNA precursors in Nagase analbuminemic rats.
Authors: Shalaby F , Shafritz D A
Published: June 2006

Based on the observation that albumin transcripts accumulate in the liver nuclear RNA fraction of Nagase analbuminemic rats (NAR), it was proposed [Esumi, H., Takahashi, Y., Sato, S., Nagase, S. & Sugimura, T. (1983) Proc. Natl. Acad. Sci. USA 80, 95-99] [corrected] that a 7-base-pair deletion at the splice donor site of intron H-I of the albumin gene in these animals leads to impaired processing of albumin pre-mRNA. To identify the specific splicing abnormality, we examined the primary structure of cytoplasmic albumin mRNA across the junctions of exons G-H-I by RNase protection mapping, Northern blot hybridization, Southern blot analysis of polymerase chain reaction-amplified cDNA, and DNA sequencing. The major albumin mRNA species in NAR showed precise deletion of exon H, suggesting that this exon was skipped during albumin pre-mRNA processing. Since the intron G-H splice donor and acceptor sites and exon H sequence are normal, the finding of exon H skipping in NAR has important implications regarding the mechanism of splice site selection. Moreover, the NAR model provides an excellent system to study splicing in vivo in a higher animal.
Superstructure of the Drosophila ribosomal gene family.
Authors: Williams S M , Robbins L G , Cluster P D , Allard R W , Strobeck C
Published: June 2006

Determining the spatial organization of middle repetitive DNA has proven difficult for several reasons. Repeated arrays are often so large that molecular methods alone cannot resolve their organization, and the lack of phenotypic markers within arrays limits the value of classical genetic analysis. We have characterized the superstructure of one repeated gene family, the ribosomal gene family of Drosophila melanogaster, by a combination of recombinational and molecular analyses of spacer-length variants. The resulting genetic maps demonstrate that some spacer variants are widely dispersed, while others are limited in their distribution. Moreover, exchange among ribosomal DNA (DNA encoding rRNA) arrays was often unequal, leading to a prediction of little or no relationship between physical location in an array and relatedness of gene family members. Extensions of our procedure may be generally useful for mapping the superstructure of repetitive DNA.
Vertical transmission of human immunodeficiency virus is correlated with the absence of high-affinity/avidity maternal antibodies to the gp120 principal neutralizing domain.
Authors: Devash Y , Calvelli T A , Wood D G , Reagan K J , Rubinstein A
Published: June 2006

Many, but not all, infants born to mothers infected with the human immunodeficiency virus (HIV) are infected in utero. We have now shown that mothers who have high-affinity/avidity antibodies directed toward the principal neutralizing domain (PND) of gp120 are less likely to transmit HIV to their children. An ELISA that preferentially measures the level of the biologically functioning, high-affinity/avidity antibodies against PND is described. In a retrospective study of 15 maternal/neonatal serum samples, the assay correctly identified the 4 uninfected and the 11 HIV-infected infants. Other clinical and laboratory parameters such as p24 antigen, phytohemagglutinin mitogenic index, and absolute surface antigen T4+ cell counts did not accurately predict HIV fetal transmission. In addition to introducing a promising diagnostic tool, this study provides the in vivo evidence that protective antibodies may prevent infection by HIV.
Molecular cloning and characterization of interferon alpha/beta response element binding factors of the murine (2'-5')oligoadenylate synthetase ME-12 gene.
Authors: Yan C , Tamm I
Published: June 2006

Seven clones encoding interferon response element binding factors have been isolated from a mouse fibroblast lambda gt11 cDNA library by using a 32P end-labeled tandem trimer of the mouse (2'-5')oligoadenylate synthetase gene interferon response element as a probe. Clone 16 shares strong similarity (95%) at both DNA and amino acid level with YB-1, a human major histocompatibility complex class II Y-box DNA-binding protein, and with dbpB, a human epidermal growth factor receptor gene enhancer region binding protein. The product of the gene represented by clone 16 may represent a factor that regulates multiple genes by binding to a variety of 5' regulatory elements. Clone 25 is a 2407-base-pair-long cDNA and contains a putative 311-amino acid open reading frame corresponding to an estimated mass of 35.5 kDa. This putative protein, designated as interferon response element binding factor 1 (IREBF-1), contains an acidic domain, three heptad repeat leucine arrays, and a region that shares similarity with the yeast transcriptional factor GAL4 DNA-binding domain. Furthermore, the C terminus of IREBF-1 shows an unusual amphipathic property: within a 79-amino acid range, one side of the alpha-helical region contains a preponderance of hydrophobic amino acids and the other side contains hydrophilic amino acids. This type of structure provides a strong hydrophobic force for protein-protein interaction.
Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects.
Authors: Mãrtensson J , Meister A , Mrtensson J
Published: June 2006

Glutathione deficiency in newborn rats, produced by administration of L-buthionine-(S,R)-sulfoximine, a transition-state inactivator of gamma-glutamylcysteine synthetase, decreases ascorbate levels of kidney, liver, brain, and lung. These tissues, especially their mitochondria, undergo severe damage and the animals die within a few days. When glutathione levels are markedly decreased, ascorbate levels decrease leading to formation of dehydroascorbate, which is degraded. Ascorbate has high antioxidant activity, but it (and other antioxidants such as alpha-tocopherol) must be maintained in reduced forms. These studies show in vivo that an important function of glutathione is to maintain tissue ascorbate. Administration of large doses of ascorbate (but not of dehydroascorbate) to buthionine sulfoximine-treated newborn rats decreases mortality, leads to normal levels of ascorbate, and spares glutathione. Newborn rats given lower doses of buthionine sulfoximine develop cataracts that, as shown previously, can be prevented by giving glutathione monoester; as found here, such cataracts can be partially prevented by administration of high doses of ascorbate or dehydroascorbate. Ascorbate spares glutathione indicating that these compounds have similar antioxidant actions. Ascorbate may have reductive functions that are not efficiently performed by glutathione. Although glutathione normally functions to maintain ascorbate, alpha-tocopherol, and other cellular components in reduced states, ascorbate can serve as an essential antioxidant in the presence of severe glutathione deficiency.
Cloning and expression of the human vasoactive intestinal peptide receptor.
Authors: Sreedharan S P , Robichon A , Peterson K E , Goetzl E J
Published: June 2006

Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands.
Kinetics of expression of multiply spliced RNA in early human immunodeficiency virus type 1 infection of lymphocytes and monocytes.
Authors: Klotman M E , Kim S , Buchbinder A , DeRossi A , Baltimore D , Wong-Staal F
Published: June 2006

The genome of human immunodeficiency virus type 1 (HIV-1) encodes at least six proteins involved in regulation as well as the structural proteins Gag, Pol, and Env. The interplay of the various regulators generates early and late transcriptional phases in the HIV-1 life cycle; the earliest RNA is enriched in subgenomic species, and the genomic transcript appears at the later stage of infection. We investigated the nature of the mRNAs expressed in the early stages of infection when the 2 kilobase subgenomic species predominate. RNA was analyzed in the early phase of a one-step growth cycle of HIV-1 infection in T-lymphoid and monocytic cell lines by using PCR amplification of in vitro-synthesized viral cDNAs. In both cell lines, expression of Tat-, Rev-, and Nef-specific messages appeared simultaneously and could be detected within 8-12 hr of infection but in different amounts with a predominance of Nef-specific message. The Env-specific message could be detected as early as the Rev-specific message, indicating that expression of at least small amounts of the singly spliced message could occur before the accumulation of Rev.
Relationship of genes encoding Ca2+/calmodulin-dependent protein kinase Gr and calspermin: a gene within a gene.
Authors: Ohmstede C A , Bland M M , Merrill B M , Sahyoun N
Published: June 2006

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells (CaM kinase Gr) is a neuronal calmodulin-dependent protein kinase whose purification and partial cloning from rat brain has been described. A combination of the polymerase chain reaction and cDNA library screening was used to determine the DNA sequence that encodes most of the remaining polypeptide sequence. The deduced amino acid sequence was confirmed by comparison with the peptide sequence from purified CaM kinase Gr. Analysis of this sequence indicated the presence of potential catalytic, regulatory, and association domains with 42% overall homology to the alpha subunit of another neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase II. The degree of homology within the catalytic domain was 58% with conservation of all invariant amino acids. The portion of sequence that extended from the hypothesized calmodulin-binding domain to the carboxyl terminus of the protein was identical at both the amino acid and nucleotide level to the noncatalytic, calmodulin-binding protein calspermin from rat testis. Screening a genomic library with a portion of the cDNA for CaM kinase Gr allowed the isolation of a genomic clone that contained at least 9 kilobases (kb) of the gene for CaM kinase Gr. Analysis of the sequence revealed that the coding sequences for calspermin were contained within the CaM kinase Gr gene and that alternative splicing of internal exons may lead to the formation of the two different proteins, CaM kinase Gr and calspermin.
Formation of the tetraploid intermediate is associated with the development of cells with more than four centrioles in the elastase-simian virus 40 tumor antigen transgenic mouse model of pancreatic cancer.
Authors: Levine D S , Sanchez C A , Rabinovitch P S , Reid B J
Published: June 2006

The development of pancreatic cancer in transgenic mice expressing the simian virus 40 tumor antigen placed under controlling regions of the elastase I gene is characterized by the sequential appearance of tetraploid and then multiple aneuploid cell populations. Pancreatic tissues from such transgenic mice were studied between 8 and 32 days of age. Virtually 100% of acinar cell nuclei had immunohistochemically detectable tumor antigen by 18 days. Tetraploid cells were demonstrated by DNA content flow cytometry by 20 days and were associated with the appearance of interphase cells that had 5-11 centrioles per cell in single thin sections of pancreatic tissue examined by electron microscopy. Mitotic cells also were observed that had 5 or more centrioles per cell that were incorporated into the poles of bipolar or at least tripolar spindle apparatuses. These observations indicate that formation of the tetraploid intermediate in the diploid----tetraploid----aneuploid sequence of pancreatic tumor formation in elastase-simian virus 40 tumor antigen transgenic mice is accompanied by the development of cells with 5 or more centrioles that can be incorporated into the poles of abnormal mitotic spindles. We speculate that cells with more than 4 centrioles are predisposed to the formation of multipolar mitoses that may yield daughter cells with chromosomal gains and losses, resulting in the subsequent development of aneuploid tumors.
Micromotion of mammalian cells measured electrically.
Authors: Giaever I , Keese C R
Published: June 2006

Motility is a fundamental property of mammalian cells that normally is observed in tissue culture by time lapse microscopy where resolution is limited by the wavelength of light. This paper examines a powerful electrical technique by which cell motion is quantitatively measured at the nanometer level. In this method, the cells are cultured on small evaporated gold electrodes carrying weak ac currents. A large change in the measured electrical impedance of the electrodes is observed when cells attach and spread on these electrodes. When the impedance is tracked as a function of time, fluctuations are observed that are a direct measure of cell motion. Surprisingly, these fluctuations continue even when the cell layer becomes confluent. By comparing the measured impedance with a theoretical model, it is clear that under these circumstances the average motions of the cell layer of 1 nm can be inferred from the measurements. We refer to this aspect of cell motility as micromotion.
Molecular cloning of a gene encoding the histamine H2 receptor.
Authors: Gantz I , Schäffer M , DelValle J , Logsdon C , Campbell V , Uhler M , Yamada T
Published: June 2006

The H2 subclass of histamine receptors mediates gastric acid secretion, and antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the gastrointestinal tract. The physiological action of histamine has been shown to be mediated via a guanine nucleotide-binding protein linked to adenylate cyclase activation and cellular cAMP generation. We capitalized on the technique of polymerase chain reaction, using degenerate oligonucleotide primers based on the known homology between cellular receptors linked to guanine nucleotide-binding proteins to obtain a partial-length clone from canine gastric parietal cell cDNA. This clone was used to obtain a full-length receptor gene from a canine genomic library. Histamine increased in a dose-dependent manner cellular cAMP content in L cells permanently transfected with this gene, and preincubation of the cells with the H2-selective antagonist cimetidine shifted the dose-response curve to the right. Cimetidine inhibited the binding of the radiolabeled H2 receptor-selective ligand [methyl-3H]tiotidine to the transfected cells in a dose-dependent fashion, but the H1-selective antagonist diphenhydramine did not. These data indicate that we have cloned a gene that encodes the H2 subclass of histamine receptors.
Alanine scanning site-directed mutagenesis of the zinc fingers of transcription factor ADR1: residues that contact DNA and that transactivate.
Authors: Thukral S K , Morrison M L , Young E T
Published: June 2006

To identify functionally important amino acids in the two zinc fingers of transcription factor ADR1 [alcohol dehydrogenase (ADH) II synthesis regulator], oligonucleotide-directed mutagenesis was used to substitute alanine for the original amino acid at each position in both fingers. The effects of these mutations on DNA binding and thermal stability of ADR1 in vitro and on activation of ADH2 expression in vivo were measured. The DNA binding activity was remarkably heatstable. Amino acids that are candidates for DNA contact sites were identified in the finger-tip and alpha-helical region of each finger, three in the first finger and two in the second. Unexpectedly, an acidic residue in the first finger was essential for transactivation, but its replacement by alanine had no effect on DNA binding. Substitution at several highly conserved positions did not affect ADR1 functions. The ADR1 zinc fingers appear to make relatively few energetically significant contacts to DNA, perhaps as few as three in the first finger and one in the second.
Magnetospheric imaging with low-energy neutral atoms.
Authors: McComas D J , Barraclough B L , Elphic R C , Funsten H O , Thomsen M F
Published: June 2006

Global imaging of the magnetospheric charged particle population can be achieved by remote measurement of the neutral atoms produced when magnetospheric ions undergo charge exchange with cold exospheric neutral atoms. Previously suggested energetic neutral atom imagers were only able to measure neutral atoms with energies typically greater than several tens of keV. A laboratory prototype has been built and tested for a different type of space plasma neutral imaging instrument, which allows neutral atoms to be imaged down to <1 keV. Such low-energy measurements provide greater sensitivity for imaging the terrestrial magnetosphere and allow the bulk of the magnetospheric ion distribution, typically centered below 10 keV, to be observed rather than just the high-energy tail of the distribution. The low-energy neutral atom measurements are made possible by utilizing charge state modifications that occur when an initially neutral atom passes through an ultrathin carbon foil. Oxygen, for example, is highly electronegative, and for energies of approximately 10-30 keV, the O- yield is approximately 30%, essentially independent of the charge state of the incident oxygen atom. These ions are energy per charge analyzed, and the UV background is rejected by using an electrostatic analyzer. Imaging of other ion species, such as hydrogen, could also be accomplished by using ultrathin foil-induced charge state modifications. The technique described in this paper provides a method for imaging charge exchange neutrals from the terrestrial magnetosphere and would also have applications for similar imaging in other planetary or cometary environs. The Inner Magnetosphere Imaging Mission, which the National Aeronautics and Space Administration is presently considering, would provide a nearly ideal platform for low-energy neutral atom imaging, and such measurements would substantially enhance the scientific yield of this mission.
Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes.
Authors: Giovannelli A , Grassi F , Mattei E , Mileo A M , Eusebi F , Giovanelli A
Published: June 2006

Electrophysiological, biochemical, and Ca2+ imaging studies of cultured mouse myotubes were used to investigate whether the neurotransmitter acetylcholine causes an increase in intracellular Ca2+ concentration ([Ca2+]i) through activation of a second messenger system. Bath applications of acetylcholine to myotubes (i) elicited a significant membrane current even in a Na(+)-free Ca2+ medium, when the current was carried mainly by calcium ions; (ii) caused a rapid and transient cytosolic accumulation of inositol 1,4,5-trisphosphate; (iii) evoked a conspicuous alpha-bungarotoxin-sensitive long-lasting [Ca2+]i enhancement even in the presence of Cd2+; and (iv) transiently increased [Ca2+]i when cells were equilibrated in a Ca(2+)-free atropine-containing medium. We propose that, in addition to opening ion channels, the nicotinic action of acetylcholine on the muscle cell membrane increases [Ca2+]i through activation of the inositol 1,4,5-trisphosphate second messenger system and mobilization of Ca2+ from intracellular stores.