bluuebunny/crossref_metadata_2025 huggingface dataset:
Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences.
Authors: Vilgalys R , Sun B L
Published: June 2006

Evidence from molecular systematic studies suggests that many mushroom species may be quite ancient. Gene phylogenies were developed to examine the relationship between reproductive isolation, genetic divergence, and biogeography in oyster mushrooms (Pleurotus). Sequence data were obtained for two regions of DNA from populations belonging to eight intersterility groups (biological species). Phylogenetic analysis of sequences from the 5' portion of the nuclear encoded large subunit rDNA demonstrates an ancient origin for four intersterility groups of broad geographic distribution (world-wide), with a more recent radiation of several intersterility groups that are restricted to the Northern Hemisphere. An expanded analysis using sequence data from the more variable rDNA internal transcribed spacer region also reveals a phylogenetically based pattern of genetic divergence associated with allopatric speciation among populations from different continents in the Northern Hemisphere. The ability of rDNA sequences to resolve phylogenetic relationships among geographically isolated populations within intersterility groups illustrates the importance of biogeography for understanding speciation in Pleurotus. Patterns of geographic distribution among intersterility groups suggest that several species lineages evolved quite early, with recently evolved groups restricted to the Northern Hemisphere and older lineages occurring throughout the world. Based on phylogenetic evidence, analysis of historical biogeography using area cladograms shows that multiple dispersal and vicariance events are responsible for patterns of speciation observed.
DNA sequence support for a close phylogenetic relationship between some storks and New World vultures.
Authors: Avise J C , Nelson W S , Sibley C G
Published: June 2006

Nucleotide sequences from the mitochondrial cytochrome b gene were used to address a controversial suggestion that New World vultures are related more closely to storks than to Old World vultures. Phylogenetic analyses of 1-kb sequences from 18 relevant avian species indicate that the similarities in morphology and behavior between New World and Old World vultures probably manifest convergent adaptations associated with carrion-feeding, rather than propinquity of descent. Direct sequence evidence for a close phylogenetic alliance between at least some New World vultures and storks lends support to conclusions reached previously from DNA.DNA hybridization methods and detailed morphology-based appraisals, and it illustrates how mistaken assumptions of homology for organismal adaptations can compromise biological classifications. However, there was a lack of significant resolution for most other branches in the cytochrome b phylogenetic reconstructions. This irresolution is most likely attributable to a close temporal clustering of nodes, rather than to ceiling effects (mutational saturation) producing an inappropriate window of resolution for the cytochrome b sequences.
A synthetic retinoid antagonist inhibits the human immunodeficiency virus type 1 promoter.
Authors: Lee M O , Hobbs P D , Zhang X K , Dawson M I , Pfahl M
Published: June 2006

Retinoids regulate a broad range of biological processes and affect cell growth and differentiation of many cell types, including the immune system. Recently, it was reported that human immunodeficiency virus type 1 (HIV-1) expression in macrophages is enhanced by retinoic acid (RA). Retinoid signals are mediated by the RA receptors (RARs) and retinoid X receptors (RXRs) that bind to specific RA responsive elements (RAREs) in the promoter region of susceptible genes. Here, we report on a RARE in the long terminal repeat (LTR) region that allows activation of the HIV-1 LTR. The RARE is composed of two consensus RARE half-sites (A/GGGTCA) arranged as a palindrome separated by 9 nucleotides and is activated by both RAR/RXR heterodimers and RXR homodimers. We show that the COUP (chicken ovalbumin upstream promoter) orphan receptors also bind to the HIV-1 RARE and repress the retinoid response of the HIV-1 RARE or the HIV-1 LTR. Furthermore, a newly discovered synthetic retinoid is shown to be a potent inhibitor of retinoid-induced activation of the HIV-1 RARE. These observations suggest additional approaches for the inhibition of HIV replication.
Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types.
Authors: Bement W M , Hasson T , Wirth J A , Cheney R E , Mooseker M S
Published: June 2006

Myosin diversity in the human epithelial cell line Caco-2BBe, the porcine epithelial cell line LLC-PK1 (CL-4), human peripheral blood leukocytes, and human liver was analyzed. PCR amplification yielded 8-11 putative myosins (depending on the cDNA source) representing six distinct myosin classes. Analysis of clones obtained by hybridization screening demonstrated that the original PCR products correspond to bona fide myosins, based on the presence of sequences highly conserved in other myosins. RNase protection analysis confirmed mRNA expression of 11 myosins in Caco-2BBe cells. Immunoblot analysis showed that at least 6 myosin immunogens are expressed in Caco-2BBe cells. The results reveal the existence of at least 11 unconventional human myosin genes, most of which are expressed in an overlapping fashion in different cell types. The abundance of myosins suggests that the myosin I vs. myosin II paradigm is inadequate to explain actin-based cellular motility.
A thyroid hormone-regulated gene in Xenopus laevis encodes a type III iodothyronine 5-deiodinase.
Authors: St Germain D L , Schwartzman R A , Croteau W , Kanamori A , Wang Z , Brown D D , Galton V A
Published: June 2006

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in the mammalian enzyme. We report here that the protein encoded by the XL-15 cDNA efficiently catalyzes the (inner ring) 5-deiodination of 3,5,3'-triiodothyronine with a Km value of 2 nM and is resistant to inhibition by propylthiouracil and aurothioglucose. Our analysis confirms that the UGA codon encodes a selenocysteine that is critical for the catalytic activity of the enzyme. In addition, the direct induction of XL-15 mRNA levels by thyroid hormone in X. laevis tadpole tail tissue and cultured cell lines correlates closely with increases in 5- (but not 5'-) deiodinase activity. These findings indicate that the XL-15 cDNA encodes a type III 5-deiodinase and underscores the importance of the trace element selenium in thyroid hormone metabolism.
Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis.
Authors: D'Souza C , Nakano M M , Zuber P
Published: June 2006

Genetic competence (the ability to internalize exogenous DNA) in Bacillus subtilis is dependent on a regulatory pathway that activates the expression of a battery of competence-specific genes. The srfA operon, encoding the subunits of surfactin synthetase, which catalyzes the nonribosomal synthesis of the peptide antibiotic surfactin, also functions in the competence regulatory pathway. The DNA encoding only one of the seven amino acid-activating domains of surfactin synthetase, the valine-activating domain (srfAB1), is necessary for competence. Deletion analysis revealed that a 569-bp fragment of srfAB1, fused to the srfA promoter, complements a srfA deletion mutation (delta srfA) with respect to competence. This fragment contains an open reading frame consisting of 46 amino acids (orf46), which is out of frame with srfAB1. A frameshift mutation in srfAB upstream of orf46 has no effect on competence but a frameshift and nonsense mutation in orf46 resulted in failure to complement the delt srfA mutation. These results indicate that orf46 encodes the srfA-associated competence regulatory factor. Computer-aided analysis of the putative orf46 product (ComS) shows similarity to the homeodomain of the POU domain class of eukaryotic transcriptional regulators.
Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.
Authors: Miller J L , Donahue R E , Sellers S E , Samulski R J , Young N S , Nienhuis A W
Published: June 2006

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.
Idiotypic mimicry and the assembly of a supramolecular structure: an anti-idiotypic antibody that mimics taxol in its tubulin-microtubule interactions.
Authors: Leu J G , Chen B X , Diamanduros A W , Erlanger B F
Published: June 2006

Taxol, originally extracted from the bark of the western yew, Taxus brevifolia, is reportedly the first of a new class of anti-cancer agents. It acts by promoting and irreversibly stabilizing microtubule assembly, thus interfering with the dynamic processes required for cell viability and multiplication. With the aim of using immunological techniques to study the mechanism of action of taxol, a monoclonal anti-idiotypic antibody that mimics taxol was prepared, using an auto-anti-idiotypic strategy. It and its Fab fragment inhibited the binding of [3H]taxol to microtubules. Moreover, like taxol, both promoted the assembly of tubulin into microtubules. These findings provide an example of an anti-idiotypic antibody capable of assembling an organized supramolecular structure from soluble cellular components. In addition, it further establishes the ability of anti-idiotypic antibodies to be functional mimics of ligand molecules bearing no structural similarity to immunoglobulins. The variable regions of the antibody have been sequenced. With the exception of the complementarity-determining region 3, the sequence of the heavy chain variable region is strikingly similar to that of an anti-idiotypic antibody raised to anti-insulin. The finding that a polypeptide can mimic taxol raises the possibility that taxol acts as a peptidomimetic compound that interferes with the function of an endogenous polypeptide.
Rapid gene-specific repair of cisplatin lesions at the human DUG/DHFR locus comprising the divergent upstream gene and dihydrofolate reductase gene during early G1 phase of the cell cycle assayed by using the exonucleolytic activity of T4 DNA polymerase.
Authors: Rampino N J , Bohr V A
Published: June 2006

A novel assay to detect strand-specific DNA repair after cellular exposure to cisplatin at IC50 levels, is used to measure rapid repair in the divergent upstream gene (DUG), a human MutS homolog, and in the bidirectional promoter for dihydrofolate reductase gene (DHFR) and the contiguous upstream DUG. Single-stranded DNA capable of hybridizing to gene-specific probes is generated enzymatically by the 3'-5' exonuclease activity of T4 DNA polymerase. The presence of cisplatin lesions inhibit the exonucleolytic activity of T4 DNA polymerase and block the formation of single-stranded DNA. This decreases the amount of complementary sequence produced when assayed by gene-specific probe hybridization. With the progression of repair, increasing quantities of single-stranded DNA become available for probe hybridization. This assay was applied to human A2780 ovarian carcinoma cells treated with cisplatin at the beginning of G1 phase. A dose-response experiment showed that the assay was applicable down to cisplatin concentrations of 2.5 microM. To assay for strand-specific gene repair, the synchronized cells were treated with cisplatin and then allowed time to repair in drug-free medium. Extensive removal of cisplatin lesions after 2 hr of cellular repair during early G1 phase in the DUG and the DUG/DHFR promoter was measured, with no evidence of repair in the unexpressed delta-globin gene. The extent of preferential DNA repair was much more distinct than has been observed previously at high-drug dosage in asynchronous cells.
The emergence of mass spectrometry in biochemical research.
Authors: Siuzdak G
Published: June 2006

The initial steps toward routinely applying mass spectrometry in the biochemical laboratory have been achieved. In the past, mass spectrometry was confined to the realm of small, relatively stable molecules; large or thermally labile molecules did not survive the desorption and ionization processes intact. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allow for the analysis of both small and large biomolecules through "mild" desorption and ionization methods. The use of ESI and MALDI mass spectrometry extends beyond simple characterization. Noncovalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery are among the areas to which ESI and MALDI mass spectrometry have been applied. This review summarizes recent developments and major contributions in mass spectrometry, focusing on the applications of MALDI and ESI mass spectrometry.
RETRACTED: Functional characterization of a nonclassical nicotine receptor associated with inositolphospholipid breakdown and mobilization of intracellular calcium pools.
Authors: Garnier M , Lamacz M , Tonon M C , Vaudry H
Published: June 2006

Classical nicotinic receptors are neurotransmitter-gated channels that, upon activation by acetylcholine, induce the opening of an intrinsic cationic channel. We have recently observed that, in frog pituitary melanotrophs, nicotine stimulates alpha-melanocyte-stimulating hormone (alpha-MSH) release through a noncholinergic mechanism. In the study reported here, we investigated the intracellular events that mediate the response of frog melanotrophs to nicotine. Nicotine was capable of stimulating alpha-MSH release in the absence of Ca2+ and/or Na+ in the extracellular medium. A short pulse of nicotine induced a rapid and transient increase of cytosolic free Ca2+ concentration ([Ca2+]i). The effect of nicotine on Ca2+ mobilization was not affected in the absence of Na+ and Ca2+ in the extracellular medium, indicating that the nicotine-evoked increase in [Ca2+]i did not result from Na+ or Ca2+ influx. Nicotine induced both an increase in inositol trisphosphate and a reduction in phosphaditylinositol bisphosphate concentrations but did not affect cAMP production. The present results indicate that nicotine-induced stimulation of alpha-MSH release in frog melanotrophs can be explained by activation of inositolphospholipid breakdown and mobilization of inositol triphosphate-dependent intracellular Ca2+ pools. These data provide evidence for the existence of an unusual type of noncholinergic nicotine receptor positively coupled to phospholipase C.
Incipient and overt stages of neoplastic transformation.
Authors: Rubin H
Published: June 2006

A subline of NIH 3T3 cells was developed that multiplies to low saturation density and produces no transformed foci in a standard assay at confluence if the cells had been kept in continuous exponential growth by passage at extremely low density. Repeated cycles of extended incubation at confluence result in the production of large dense foci with large increases in saturation density of the culture. I investigated the changes preceding overt transformation and the conditions that favor them by making a preliminary 2- or 3-week incubation of cells at high density in 2, 5, or 10% (vol/vol) calf serum (CS) followed by three consecutive assays at 2-week intervals, all in 2% CS. The cell counts on transfer of each consecutive assay constituted their saturation density, and staining of sister dishes exhibited transformed foci and other morphological evidence of transformation. To strengthen the significance of the observations, each CS category consisted of four separate but parallel lineages. The initial incubation in 2, 5, and 10% CS is referred to as the first-round (1 degree) assay. In the second-round (2 degrees) assay in 2% CS after the primary incubation for 2 weeks in different concentrations of CS, there were small but consistent increases in saturation density that were proportional to the CS concentration used in the first round. There were also large numbers of very light foci in the 2 degrees assay that increased in size as a function of the CS concentration used in the 1 degree assay but were uniform within each CS category. In the 3 degrees and 4 degrees successive assays, large dense foci appeared in a sporadic manner but these were also dependent on the CS concentration in the initial incubation. Cultures kept in the 1 degree assay in 5 and 10% CS for 3 weeks produced large dense foci at an earlier stage of the subsequent assays than did those kept for only 2 weeks in the 1 degree assay. The barely perceptible nature of the early morphological changes suggests an analogy to incipient neoplasia. Their graded nature, relatively high frequency, regularity of occurrence, and uniformity of appearance are characteristic of an epigenetic process, which Foulds [Foulds, L. (1969) Neoplastic Development, Vol. I (Academic, New York), preface, pp. 41-89] cited as the most plausible basis for incipient neoplasia [corrected]. In contrast, the low-frequency and sporadic occurrence of the large dense foci, which have the main features of overt neoplasia, are consistent with both genetic and epigenetic processes.
Two separate areas of the brain differentially guide the development of a song control nucleus in the zebra finch.
Authors: Akutagawa E , Konishi M
Published: June 2006

A brain nucleus that is important for the generation of song in the adult male zebra finch (Poephila guttata), the robust nucleus of the archistriatum (RA), receives dual inputs from two other telencephalic song nuclei: the hyperstriatum ventrale pars caudale (HVc) and the lateral magnocellular nucleus of the anterior neostriatum (L-MAN). We lesioned each of these afferent inputs to the RA early in development, either by themselves or both at the same time in the same side of the brain, to determine what influences each of these nuclei exerts on the normal development of the RA into adulthood. We found that lesioning the HVc in a 20-day-old male zebra finch prevents the later increase in RA volume and soma size that would normally occur around 35 days post-hatching. MAN lesions at this same early age, on the other hand, had a large effect on reducing the volume and cell number of RA neurons, without affecting soma size. Lesioning both inputs early in development induced considerable RA neuronal cell death and atrophy of the nucleus. This study shows that the development of the RA is affected differently by each of its two input nuclei.
Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region.
Authors: Stevens A M , Dolan K M , Greenberg E P
Published: June 2006

LuxR, the Vibrio fischeri luminescence gene (lux) activator, is the best-studied member of a family of bacterial transcription factors required for cell density-dependent expression of specific genes involved in associations with eukaryotic hosts. Neither LuxR nor any other LuxR homolog has been shown to bind DNA directly. We have purified the LuxR C-terminal transcriptional activator domain from extracts of recombinant Escherichia coli in which this polypeptide was expressed. The purified polypeptide by itself binds to lux regulatory DNA upstream of the lux box, a 20-bp palindrome that is required for LuxR activity in vivo, but it does not bind to the lux box. However, the LuxR C-terminal domain together with RNA polymerase protects a region including the lux box and the lux operon promoter from DNase I cleavage. There is very little protection of the lux operon promoter region from DNase I digestion in the presence of RNA polymerase alone. Apparently, there is a synergistic binding of the LuxR C-terminal domain and RNA polymerase to the promoter region. The upstream binding region for the purified polypeptide encompasses a binding site for cAMP receptor protein (CRP). Under some conditions, CRP binding can block the binding of the LuxR C-terminal domain to the upstream binding region, and it can also block the synergistic binding of the LuxR C-terminal domain and RNA polymerase to the lux box and luminescence gene promoter region. This description of DNA binding by the LuxR C-terminal domain should lead to an understanding of the molecular interactions of the LuxR family of transcriptional activators with regulatory DNA.
Molecular cloning and derived primary structure of cobra venom factor.
Authors: Fritzinger D C , Bredehorst R , Vogel C W
Published: June 2006

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. Like C3b, CVF forms with factor B and factor D in human and mammalian serum the bimolecular C3/C5 convertase. This functional similarity of CVF and C3 correlates with many structural similarities, which led to the suggestion that CVF is evolutionally related to C3. We report here the molecular cloning and derived primary structure of CVF. CVF mRNA is > 5924 nucleotides in length. It contains a single open reading frame of 4926 nucleotides, coding for a pre-pro-protein of 1642 amino acids. The deduced amino acid sequence reveals approximately 70% protein similarity to mammalian and human C3 and exceeds 91% in the case of cobra C3. The single-chain pre-pro-CVF consists of a 22-amino acid signal sequence, a 627-amino acid alpha-chain, and a 989-amino acid precursor chain for the CVF gamma- and beta-chains. The processing of pro-CVF involves the removal of 4 arginine residues between the alpha- and precursor chains as well as of the C3a-like and C3d-like domains from the precursor chain, thereby confirming the predicted chain homologies to C3. Pro-CVF contains five potential N-glycosylation sites, of which only three can be expected to be glycosylated in mature CVF. Like C3, pro-CVF contains 27 cysteine residues and a homologous thioester site in the C3d-like region.
Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B.
Authors: Zhang Y , Broser M , Rom W N
Published: June 2006

The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guérin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements.
Structure, characterization, and expression of the rat oxytocin receptor gene.
Authors: Rozen F , Russo C , Banville D , Zingg H H
Published: June 2006

The multiple hormonal and neurotransmitter functions of the nonapeptide oxytocin are mediated by specific oxytocin receptors (OTRs). In most target tissues, the number of OTRs is strongly regulated. Specifically, in the uterus, a dramatic OTR upregulation precedes the onset of parturition. To study the molecular mechanisms underlying OTR regulation, we have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the rat OTR gene, using sequence information derived from a human myometrial OTR cDNA. The rat OTR gene spans > 20 kb and contains three exons. A 97-bp intron is in the 5' untranslated region and a > 12-kb intron interrupts the coding region between transmembrane domains 6 and 7. The promoter region lacks an apparent TATA or CCAAT box but contains multiple putative interleukin-response elements [six NF-IL6 (C/EBP beta) and four APRF (STAT3) binding motifs], supporting the notion that interleukins may mediate labor induction via transcriptional activation of the OTR gene. The predicted amino acid sequence is 93% identical to the human OTR sequence but only 48% and 38% identical to the rat V1 and V2 vasopressin receptor sequences, respectively. At parturition, the OTR gene is highly expressed in the rat uterus and gives rise to at least three transcripts (2.9, 4.8, and 6.7 kb) which differ in the length of their 3' untranslated regions.
A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity.
Authors: Yang W S , Nevin D N , Peng R , Brunzell J D , Deeb S S
Published: June 2006

We have identified a naturally occurring mutation in the promoter of the lipoprotein lipase (LPL) gene. One of 20 patients with familial combined hyperlipidemia (FCHL) and reduced levels of postheparin plasma LPL activity was found to be a heterozygote carrier of this mutation. The mutation, a T-->C substitution at nt -39, occurred in the binding site of the transcription factor Oct-1. As a result, the transcriptional activity of the mutant promoter was < 15% of wild type, as determined by transfection studies in the human macrophage-like cell line THP-1. This decrease in promoter activity was observed in undifferentiated as well as in phorbol ester-differentiated THP-1 cells. Furthermore, the inductive effect of elevating the levels of intracellular cAMP was equally reduced. This mutation was not present among 20 FCHL patients with normal plasma LPL levels nor has it been reported among individuals with familial LPL deficiency. Thus, heterozygosity for LPL promoter mutations may be one of several factors that contribute to the etiology of FCHL.
Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes.
Authors: Bruce J L , Hubner R J , Cole E M , McDowell C I , Webster J A
Published: June 2006

To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species.
In vitro generation of hematopoietic stem cells from an embryonic stem cell line.
Authors: Palacios R , Golunski E , Samaridis J
Published: June 2006

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.
An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.
Authors: Stamnes M A , Craighead M W , Hoe M H , Lampen N , Geromanos S , Tempst P , Rothman J E
Published: June 2006

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.
12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.
Authors: Liu B , Khan W A , Hannun Y A , Timar J , Taylor J D , Lundy S , Butovich I , Honn K V
Published: June 2006

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.
Superluminal sources.
Authors: Vermeulen R C
Published: June 2006

Predictions for the apparent velocity statistics under simple beaming models are presented and compared to the observations. The potential applications for tests of unification models and for cosmology (source counts, measurements of the Hubble constant H0 and the deceleration parameter q0) are discussed. First results from a large homogeneous survey are presented. The data do not show compelling evidence for the existence of intrinsically different populations of galaxies, BL Lacertae objects, or quasars. Apparent velocities betaapp in the range 1-5 h-1, where h = H0/100 km.s-1.Mpc-1 [1 megaparsec (Mpc) = 3.09 x 10(22) m], occur with roughly equal frequency; higher values, up to betaapp = 10 h-1, are rather more scarce than appeared to be the case from earlier work, which evidently concentrated on sources that are not representative of the general population. The betaapp distribution suggests that there might be a skewed distribution of Lorentz factors over the sample, with a peak at gammab approximately 2 h-1 and a tail up to at least gammab approximately 10 h-1. There appears to be a clearly rising upper envelope to the betaapp distribution when plotted as a function of observed 5-GHz luminosity; a combination of source counts and the apparent velocity statistics in a larger sample could provide much insight into the properties of radio jet sources.
Identification of an inducible surface molecule specific to fusing macrophages.
Authors: Saginario C , Qian H Y , Vignery A
Published: June 2006

Multinucleated giant cells and osteoclasts arise through the fusion of mononuclear phagocyte precursors. To elucidate the mechanism by which cells of monocytic lineage fuse and differentiate into giant cells and osteoclasts, we hypothesized that, as with other cell fusion events, specific surface molecules mediate the adhesion/fusion process. It has been observed that macrophages can be induced to fuse with one another in response to specific stimuli or when placed in a specific microenvironment. The formation of giant cells is primarily associated with chronic inflammatory reactions and tumors, while osteoclasts differentiate on bone which they resorb. The fact that, under normal conditions, macrophages and monocytes fail to fuse in regions and tissues where they are present in large numbers suggests the regulated and transient expression of potential fusion molecules. To identify such a fusion-associated molecule, we established a macrophage fusion assay and generated monoclonal antibodies (mAbs) that alter the fusion of macrophages in vitro. We selected four mAbs that each had the ability to block the fusion but not the aggregation of macrophages in vitro. All four antibodies recognize surface proteins of 150 kDa. The expression of the antigens recognized by all four mAbs is restricted to macrophages that have been induced to fuse in vitro and in vivo and is inducible, transient, and regulated, as neither nonfusing macrophages nor macrophages fused in vitro express these antigens. These results support the hypothesis that macrophage fusion is mediated by specific fusion/adhesion molecules and also provide a means to study the molecular mechanisms of macrophage fusion.
Functional chicken gizzard heavy meromyosin expression in and purification from baculovirus-infected insect cells.
Authors: Onishi H , Maéda K , Maéda Y , Inoue A , Fujiwara K
Published: June 2006

The heavy chain and the essential and the regulatory light chains of chicken gizzard heavy meromyosin (HMM) were coexpressed in Spodoptera frugiperda (fall armyworm) cells infected with a mixture of two recombinant Autographa californica baculoviruses. Soluble HMM consisting of the heavy chain and the two types of light chains was obtained. The recombinant HMM was purified from the virus-infected cells and characterized. The regulatory light chain of the isolated recombinant HMM was phosphorylated by myosin light chain kinase in the presence of calmodulin in a Ca(2+)-dependent manner. The ATPase of the recombinant HMM was activated by rabbit skeletal muscle actin when myosin light chain kinase, calmodulin, and Ca2+ were present in the reaction medium. Chicken gizzard tropomyosin enhanced the actin-activated ATPase activity. The recombinant HMM decorated actin filaments, displaying the characteristic arrowhead pattern along the filaments. This report on a functional recombinant double-headed smooth muscle myosin fragment opens the way to detailed studies on the molecule.
The leukemic core binding factor beta-smooth muscle myosin heavy chain (CBF beta-SMMHC) chimeric protein requires both CBF beta and myosin heavy chain domains for transformation of NIH 3T3 cells.
Authors: Hajra A , Liu P P , Wang Q , Kelley C A , Stacy T , Adelstein R S , Speck N A , Collins F S
Published: June 2006

An inversion of chromosome 16 associated with the M4Eo subtype of acute myeloid leukemia produces a chimeric protein fusing the beta subunit of the transcription factor core binding factor (CBF beta) to the tail region of smooth muscle myosin heavy chain (SMMHC). We investigated the oncogenic properties of this CBF beta-SMMHC chimeric protein using a 3T3 transformation assay. NIH 3T3 cells expressing CBF beta-SMMHC acquired a transformed phenotype, as indicated by their ability to form foci, grow in soft agarose, and form tumors in nude mice. Cells expressing normal CBF beta or the SMMHC tail domain did not become transformed. Electrophoretic mobility-shift assays showed that extracts from cells transformed by CBF beta-SMMHC no longer formed the normal CBF/DNA complex but instead formed a much larger complex that did not migrate into the gel. Analysis of CBF beta-SMMHC deletion mutants demonstrated that the chimeric protein was transforming only if two domains were both present: (i) CBF beta sequences necessary for association with the CBF alpha subunit, and (ii) SMMHC sequences important for the formation of multimeric filaments. These results are direct evidence that CBF beta-SMMHC can function as an oncoprotein.
A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase.
Authors: Huibregtse J M , Scheffner M , Beaudenon S , Howley P M
Published: June 2006

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).
Endogenously opsonized particles divert prostanoid action from lethal to protective in models of experimental endotoxemia.
Authors: Eierman D F , Yagami M , Erme S M , Minchey S R , Harmon P A , Pratt K J , Janoff A S
Published: June 2006

We report that, in rats, the lethal consequences of high-dose endotoxin challenge are exacerbated by the intravascular administration of prostaglandin E1 but attenuated by the intravascular administration of endocytosable particles. This protection is mediated by opsonins. Nonopsonizable particles were unable to provide protection unless first pseudoopsonized with antibody directed against the CR3 (CD11b/CD18) phagocyte receptor. We show that endogenously opsonized particles can act in concert with prostaglandin E1 (putatively by elevation of neutrophil intracellular cAMP and the resultant downregulation of CR3) to completely rescue animals from the lethal late-stage sequelae of experimental endotoxemia. These data illustrate that the interaction of particles with cellular receptors can transform the overall systemic response to prostaglandin E1 from pro- to antiinflammatory. This suggests a role for multiple receptor engagement events in defining the systemic prostaglandin response and offers a rationale for developing new therapeutic modalities in the treatment of sepsis and other inflammatory diseases.
'White Flight'? The Production and Reproduction of Immigrant Concentration Areas in Swedish Cities, 1990-2000
Authors: Bråmå Åsa
Published: June 2006

The article investigates whether processes similar to 'White flight' and 'White avoidance', known from American research on residential segregation, have played a role in the increased concentration of immigrants that has affected many residential areas in Swedish cities during the 1990s. By means of a comprehensive and unique dataset, processes of neighbourhood transition and mobility are described and analysed for a selection of residential areas that have experienced increased immigrant concentration during the 1990s. The results show that 'Swedish avoidance', i.e. low in-migration rates among Swedes, rather than 'Swedish flight', i.e. high out-migration rates, has been the main driving-force behind the production and reproduction of immigrant concentration areas.
Household Savings and Residential Mobility in Informal Settlements in Bhopal, India
Authors: Lall Somik V. , Suri Ajay , Deichmann Uwe
Published: June 2006

Strategies to address the problem of informal settlements have focused on slum upgrading, sites-and-services programmes and tenure security. There has been less attention on what enables slum-dwellers to transition into the formal housing sector without direct intervention. This paper investigates residential mobility among slum-dwellers in Bhopal, India. One in five households succeeds in leaving a slum settlement and a major determinant is the ability to save on a regular basis. Due to limited outreach of institutional housing finance, most slum-dwellers rely solely on household savings for purchasing a house. These findings underscore the urgent need to improve savings instruments for slum-dwellers and to downmarket housing finance to reach the poorest residents of rapidly growing cities in developing countries.
Spatial Dynamics of the Housing Market: An Interurban Perspective
Authors: Jones Colin , Leishman Chris
Published: June 2006

A major unproven hypothesis in housing economics is that regional ripple effects are caused by household migration between regions. This paper examines household migration and price ripples at the level of local housing markets driven partly by the fact that such migration linkages are likely to be more pronounced between local, rather than regional, housing markets. Following a review of regional housing market studies, several hypotheses linking the existence of lead-lag relationships and cointegration with the scale of migratory linkages between local housing market areas (LHMAs) are proposed. Using private housing transactions data for Strathclyde, a sub-region of Scotland, the paper identifies two clusters of LHMAs that differ in terms of their migratory linkages with Glasgow, the hypothesised leading housing market of the sub-region. Tests for lead-lag relationships and cointegration confirm the link between migration and house price changes.
A Continental Ambience? Lessons in Managing Alcohol-related Evening and Night-time Entertainment from Four European Capitals
Authors: Roberts Marion , Turner Chris , Greenfield Steve , Osborn Guy
Published: June 2006

Attempts to revitalise the centres of British towns and cities in the 1990s drew on the concept of the 24-hour city and, by extension, into liquor licensing reform. The protagonists for the 24-hour city, and many magistrates and local authorities, assumed that a relaxation of British licensing laws would bring about a more civilised mode of alcohol consumption and deliver a 'continental ambience' to urban life that would extend into the night. This paper brings forward evidence from a cross-cultural comparison of four European cities to demonstrate that a 'continental' style of alcohol consumption is supported by a variety of controls and enforcement measures. It concludes that British free market attitudes to licensing reform will undermine the government's professed aspirations for an 'urban renaissance' of cultural inclusion and animation.