bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.
Authors: Matijasevic Z , Boosalis M , Mackay W , Samson L , Ludlum D B
Published: May 2006

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.
Genes in one megabase of the HLA class I region.
Authors: Wei H , Fan W F , Xu H , Parimoo S , Shukla H , Chaplin D D , Weissman S M
Published: May 2006

To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex. These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F. In addition to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by us and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region. We also identified a long repetitive DNA element in the region between HLA-B and HLA-E. Homologues of this element were located at several sites in the human genome outside of the HLA complex. The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions. Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.
A masculinizing effect on the auditory systems of human females having male co-twins.
Authors: McFadden D
Published: May 2006

Spontaneous otoacoustic emissions (SOAEs) are continuous, essentially tonal sounds that are produced by many normal-hearing cochleas. In humans, females generally exhibit more SOAEs than males, a sex difference that exists from birth. However, it is shown here that females having male co-twins [opposite-sex dizygotic (OSDZ) females] exhibit about half the average number of SOAEs per ear observed in same-sex female twins or female non-twins. Indeed, the average in OSDZ females is about the same as that seen in males--twins or non-twins. The explanation offered here is that prenatal exposure to high levels of androgens has produced a masculinizing effect on the auditory systems of these OSDZ females. Prenatal masculinizing effects have long been recognized in certain litter-bearing mammals, but their existence in humans is not well-studied.
Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology.
Authors: Rothenberg B E , Hayes B K , Toomre D , Manzi A E , Varki A
Published: May 2006

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efficiency. The resulting adducts show excellent fractionation by reverse-phase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of defined structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries.
Distortion of area and dimension under quasiconformal mappings in the plane.
Authors: Astala K
Published: May 2006

We find the exact estimates for the distortion of area and Hausdorff dimension under a K-quasiconformal mapping of the complex plane. This solves also the problem of finding the precise bound p(K) of the exponents p such that for each planar K-quasiconformal mapping f the Jacobian Jf is locally p-integrable; it follows that p(K) = 2K/(K - 1). Further consequences include among others the regularity and removability results for quasiregular mappings and sharp estimates for the complex Hilbert transform.
The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity.
Authors: King C Y , Weiss M A
Published: May 2006

SRY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome, regulates a genetic switch in male development. Impairment of this switch leads to intersex abnormalities of the newborn and is observed in association with mutations in the SRY DNA-binding domain [the high-mobility-group (HMG) box]. Here we show that the SRY HMG box exhibits a novel mechanism of DNA recognition: partial intercalation of a nonpolar side chain in the DNA minor groove. Base stacking (but not base pairing) is interrupted at the site of insertion. Sequence specificity reflects topological requirements of partial intercalation rather than direct readout of base-specific functional groups. Our results predict that the SRY HMG box inserts an alpha-helix into a widened minor groove at the center of a sharp DNA bend. A similar mechanism may underlie binding of SRY and homologous HMG proteins to four-way junctions (Holliday intermediates) and other noncanonical DNA structures.
Allele-specific peptide ligand motifs of HLA-C molecules.
Authors: Falk K , Rötzschke O , Grahovac B , Schendel D , Stevanović S , Gnau V , Jung G , Strominger J L , Rammensee H G
Published: May 2006

The consensus motifs of HLA-Cw3, -Cw4, -Cw6, and -Cw7 ligands were determined by pool sequencing. Together with information obtained by sequencing of some prominent individual peptides, the results indicate the following: (i) all four HLA-C molecules are associated with peptides. (ii) These peptides adhere to allele-specific motifs that are similar to those of to HLA-A or -B molecules; they have a preferred length of nine amino acids and an anchor residue at the C terminus. (iii) All four HLA-C molecules analyzed exhibit related peptide motifs, although each allelic product shows individual characteristics in fine specificity. (iv) Processing and origin of peptides appear not to be different from that of other class I molecules. (v) No obvious difference at C-terminal position 9 was present in the peptides isolated from the two dimorphic variants of HLA-C that determine dominant resistance to natural killer NK1-specific cells (HLA-Cw4, -Cw6) or to NK2-specific cells (HLA-Cw3, -Cw7) and that differ in two residues in or near the pocket at position 9.
Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli.
Authors: Dailey F E , Berg H C
Published: May 2006

We report the isolation and characterization of Escherichia coli mutants (dsbB) that fail to assemble functional flagella unless cystine is present. Flagellar basal bodies obtained from these mutants are missing the L and P rings. This defect in assembly appears to result from an inability to form a disulfide bond in the P-ring protein (FlgI). Cystine suppresses this defect in dsbB strains. We also show that dsbA strains [Bardwell, J. C. A., McGovern, K. & Beckwith, J. (1991) Cell 67, 581-589] fail to assemble P rings, apparently from a similar failure in disulfide bond formation. However, cystine does not completely suppress this defect in dsbA strains. Thus, disulfide bond formation in FlgI is essential for assembly. DsbA likely puts in that bond directly, whereas the DsbB product(s) play a role in oxidizing DsbA, so that it can be active.
SRY recognizes conserved DNA sites in sex-specific promoters.
Authors: Haqq C M , King C Y , Donahoe P K , Weiss M A
Published: May 2006

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a single-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by high-mobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Mullerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation. Our results support the hypothesis that SRY directly controls male development through sequence-specific regulation of target genes.
Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.
Authors: Vihko P , Kurkela R , Porvari K , Herrala A , Lindfors A , Lindqvist Y , Schneider G
Published: May 2006

Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.
High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.
Authors: Svab Z , Maliga P
Published: May 2006

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
Cytolocalization of zeatin O-xylosyltransferase in Phaseolus.
Authors: Martin R C , Mok M C , Mok D W
Published: May 2006

Zeatin O-xylosyltransferase (EC 2.4.2.-) mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose in immature seeds of Phaseolus vulgaris. Tissue printing with a monoclonal antibody specific for the enzyme and a cDNA probe demonstrated that the enzyme was primarily localized and synthesized in the endosperm. Immunolocalization performed on monolayer endosperm at the free-nuclei stage and on EM sections demonstrated that the enzyme was associated with the nucleus as well as with the cytoplasm. Immunoanalysis of nuclear fractions revealed that the enzyme was retained in the nuclear pellet. Western analysis also showed that the enzyme was present in the nuclei of cotyledons and endosperm callus. The findings suggest that the enzyme may be involved in the nuclear-cytoplasmic transport of cytokinins and related molecules or, possibly, with chromatin of rapidly dividing cells.
A protein factor for ras p21-dependent activation of mitogen-activated protein (MAP) kinase through MAP kinase kinase.
Authors: Itoh T , Kaibuchi K , Masuda T , Yamamoto T , Matsuura Y , Maeda A , Shimizu K , Takai Y
Published: May 2006

To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.
Effects of hybridization and selection on floral isolation.
Authors: Grant V
Published: May 2006

This paper examines the case of natural hybridization between two angiosperm species (A and B) which are mechanically and ethologically isolated (or florally isolated). What is the effect of hybridization on the pollination system, and conversely, what is the effect of the pollinators on the outcome of the hybridization? The original floral isolation is based on an interspecific differentiation in floral characters, the floral mechanisms of the parental species being specialized for pollination by different types of pollinators with different body forms and behavioral traits. F1 hybrids of A x B have intermediate floral characters and serve as bridges for interspecific cross-pollination. The problem next shifts to the changes in floral characters and mode of pollination that are likely to occur in later generations in the hybrid population. The pollinators of species A and species B exert selective pressures on the hybrid population. If the normal pollinator of A is more abundant, active, and effective in the hybrid population than the pollinator of B, selection is expected to favor a reversion toward the floral characters and pollination system of A. The opposite condition, in which the pollinator of B is predominant, leads to the reciprocal result, reversion toward B. If the two types of pollinators are more or less the same in number of flower visits and pollination effectiveness, their combined selective pressure should produce later-generation derivatives with intermediate floral characters suited for both pollinators. Three western North American plant groups containing florally isolated species that hybridize (Ipomopsis, Diplacus, and Aquilegia) are examined in relation to these predictions. The evidence in the three groups is generally in agreement with the hypothesis.
Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue.
Authors: Lazard D , Sastre X , Frid M G , Glukhova M A , Thiery J P , Koteliansky V E
Published: May 2006

The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype.
Energetics of cooperative binding of oligonucleotides with discrete dimerization domains to DNA by triple helix formation.
Authors: Distefano M D , Dervan P B
Published: May 2006

Cooperativity in oligonucleotide-directed sequence-specific recognition of DNA by triple helix formation can be enhanced by the addition of discrete dimerization domains. The equilibrium association constants for cooperative binding of oligonucleotides that dimerize by Watson-Crick hydrogen bonds and occupy adjacent sites on double helical DNA by triple helix formation have been measured by quantitative affinity cleavage titration. For two oligonucleotides that bind unique neighboring 11-bp and 15-bp sites on double helical DNA, and dimerize by formation of an 8-bp Watson-Crick mini-helix, the free energy of binding is -8.0 and -9.7 kcal.mol-1, respectively, and the cooperative energy of interaction is -2.3 kcal.mol-1 (1 kcal = 4.18 kJ). The energetics of this artificial nucleic acid cooperative intermolecular assembly can mimic naturally occurring cooperative protein-DNA systems, such as the phage lambda repressor.
Actin is a binding protein for angiogenin.
Authors: Hu G F , Strydom D J , Fett J W , Riordan J F , Vallee B L
Published: May 2006

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor alpha, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodiimide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.
General Gittins index processes in discrete time.
Authors: El Karoui N , Karatzas I
Published: May 2006

We combine the formulation of Mandelbaum [Mandelbaum, A. (1986) Probab. Theory Rel. Fields 71, 129-147] with ideas from Whittle [Whittle, P. (1980) J. R. Stat. Soc. B 42, 143-149] to obtain a simple and constructive proof for the optimality of Gittins index processes in the general, nonmarkovian dynamic allocation (or "multi-armed bandit") problem. Our approach also provides an explicit expression for the value of this problem.
Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity.
Authors: Seeholzer S H
Published: May 2006

With 13C NMR, phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) is shown to produce mannose 6-phosphate (M6P) slowly from a much more rapidly catalyzed equilibrium between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The identity of M6P and its formation from G6P plus F6P are confirmed by 1H NMR and by the ability of PGI to convert M6P to F6P plus G6P. The possibility of contaminating phosphomannose isomerase (PMI, D-mannose-6-phosphate ketol-isomerase, EC 5.3.1.8) is ruled out by finding no exchange of the C1 proton of G6P or of M6P, whereas exchange occurs with a mixture of PMI and PGI in 2H2O. The pro-R and pro-S protons of F6P become the anomeric protons of M6P and G6P through the actions of PMI and PGI, respectively. Both isomerases exchange the C2 proton of their substrate with the medium; hence, when PGI and PMI are added together to hexose phosphate solutions in 2H2O, both the substrate and anomeric protons are exchange rapidly with deuterons from the medium. The rates of C2-epimerization of G6P and M6P by PGI are shown to be proportional to enzyme concentration and inhibited by 5-phosphoarabinoate, a competitive inhibitor of the previously demonstrated isomerase and anomerase activities of PGI. These data show that the epimerization is enzymatically catalyzed and suggest the involvement of the same active site for all three activities. A primary kinetic isotope effect of 7.5 (H/2H) on the rate constant kcat of the M6P C2-epimerase activity was determined by using a coupled enzymatic assay. A model of the mechanism of PGI is offered, which relates C2-epimerase activity to the isomerase and anomerase activities by allowing the cis-enediol intermediate to rotate about the C2-C3 bond axis followed by protonation at C2 but not at C1 from the si face.
Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts.
Authors: van Corven E J , Hordijk P L , Medema R H , Bos J L , Moolenaar W H
Published: May 2006

Some agonists of G protein-coupled receptors, such as thrombin and lysophosphatidic acid (LPA), can promote cell proliferation via a pertussis toxin (PTX)-sensitive signaling pathway. While these agonists stimulate phospholipase C and inhibit adenylate cyclase, it appears that other, as-yet-unidentified, effector pathways are required for mitogenesis. Here we report that LPA and a thrombin receptor agonist peptide rapidly activate the protooncogene product p21ras in quiescent fibroblasts. This activation is inhibited by PTX and yet not attributable to known PTX-sensitive G protein pathways, including stimulation of phospholipases, inhibition of adenylate cyclase, or modulation of ion channels. LPA- and peptide-induced p21ras activation is inhibited by the tyrosine kinase inhibitor genistein, at doses that do not affect epidermal growth factor-induced p21ras activation. Thus, a heterotrimeric G protein of the Gi subfamily regulates activation of p21ras by LPA and thrombin, possibly through an intermediary tyrosine kinase. This pathway may critically participate in mitogenic signaling downstream from certain G protein-coupled receptors.
RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template.
Authors: Telesnitsky A , Goff S P
Published: May 2006

The active sites for the polymerase and nuclease activities of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) reside in separate domains of a single polypeptide. We have studied the effects of RNase H domain mutations on DNA polymerase activity. These mutant RTs displayed decreased processivity of DNA synthesis. We also compared complexes formed between primer-templates and mutant and wild-type reverse transcriptase (RT). Although M-MuLV RT is monomeric in solution, two molecules of RT bound DNA cooperatively, suggesting that M-MuLV RT binds primer-template as a dimer. Some mutant RTs with decreased processivity failed to form the putative dimer.
Catecholaminergic neurons result from intracerebral implantation of embryonal carcinoma cells.
Authors: Wojcik B E , Nothias F , Lazar M , Jouin H , Nicolas J F , Peschanski M
Published: May 2006

A replication-defective retrovirus was used to introduce the marker gene nlsLacZ into the murine embryonal carcinoma (EC) cell line PCC7-S-aza-R-1009. Undifferentiated EC cells were implanted into the central nervous system of adult rats. One month later, the grafted cells continued to express the nlsLacZ gene. Immunohistochemical analysis demonstrated the presence of EC-derived neurons. These neurons were capable of expressing tyrosine hydroxylase and extended neurites into the host parenchyma. EC-derived glial cells could not be detected. There was no evidence of tumorigenicity. These results demonstrate the utility of EC cells for introduction of exogenous gene products into the central nervous system in experimental models of gene therapy.
ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.
Authors: Bernardi H , De Weille J R , Epelbaum J , Mourre C , Amoroso S , Slama A , Fosset M , Lazdunski M
Published: May 2006

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.
Squalamine: an aminosterol antibiotic from the shark.
Authors: Moore K S , Wehrli S , Roder H , Rogers M , Forrest J N , McCrimmon D , Zasloff M
Published: May 2006

In recent years, a variety of low molecular weight antibiotics have been isolated from diverse animal species. These agents, which include peptides, lipids, and alkaloids, exhibit antibiotic activity against environmental microbes and are thought to play a role in innate immunity. We report here the discovery of a broad-spectrum steroidal antibiotic isolated from tissues of the dogfish shark Squalus acanthias. This water-soluble antibiotic, which we have named squalamine, exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. The chemical structure of the antibiotic 3 beta-N-1-(N-[3-(4-aminobutyl)]- 1,3-diaminopropane)-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate has been determined by fast atom bombardment mass spectroscopy and NMR. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. The discovery of squalamine in the shark implicates a steroid as a potential host-defense agent in vertebrates and provides insights into the chemical design of a family of broad-spectrum antibiotics.
5' control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family.
Authors: Wade D P , Clarke J G , Lindahl G E , Liu A C , Zysow B R , Meer K , Schwartz K , Lawn R M
Published: May 2006

Elevated blood levels of apolipoprotein(a), the component of lipoprotein(a) that distinguishes it from low density lipoprotein, are a major risk factor for atherosclerosis. The apolipoprotein(a) gene is highly similar to the plasminogen gene and to at least four other genes or pseudogenes. The 5' untranslated and flanking sequences of these six genes contain extensive regions of near identity and share sequence elements involved in the initiation of transcription and translation. About 1000 base pairs of flanking DNA of each gene are sufficient to promote transcription in cultured hepatocytes. The apolipoprotein(a) gene promoter contains functional interleukin 6-responsive elements, consistent with the reported acute-phase response of apolipoprotein(a). Flanking genomic fragments of the apoliprotein(a) gene from two individuals with vastly different plasma apolipoprotein(a) concentrations have sequence differences that are reflected in differences in the rate of in vitro transcription.
Strategies for the identification of interacting proteins.
Authors: Guarente L
Published: May 2006

Many problems in modern biology involve complex arrays of interacting protein and, in some cases, RNA molecules. The initial challenge facing investigators is to identify the important players that drive the process under study. This difficult task is ameliorated somewhat by the development of methods designed to keep pace with the magnitude of this challenge. I have outlined a few of these approaches at the cutting edge of cloning interacting proteins. A perhaps more daunting prospect is to dissect the important molecules once they are in hand, to identify key interactions, and, ultimately, to move to an understanding of function in cells. For this, of course, all of the tools of genetics, biochemistry, and molecular biology, extant and yet to be developed, will have to be tapped.
Pulsed phytoplankton supply to the rocky subtidal zone: influence of internal waves.
Authors: Witman J D , Leichter J J , Genovese S J , Brooks D A
Published: May 2006

Hydrographic measurements indicate that the thermocline and the phytoplankton-rich chlorophyll maximum layer are vertically displaced over a rocky pinnacle in the central Gulf of Maine by internal waves with maximum amplitudes of 27 m. Such predictable downwelling events are linked to rapid, 2- to 3-fold increases in chlorophyll a, an indicator of phytoplankton concentration, in pulses of warm water recorded 4 cm above the bottom (29-m depth). The 1.5-5.6 degrees C temperature fluctuations had an average period of 10.6 min and were generated on both ebb and flood tides. Local lee waves and the arrival of solitons propagated from Georges Bank are hypothesized to explain the timing of the internal waves. Because internal waves and chlorophyll maxima are pervasive features of stratified temperate seas, this mechanism of food coupling should be common in other rocky subtidal habitats.
Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.
Authors: Molineaux S M , Casano F J , Rolando A M , Peterson E P , Limjuco G , Chin J , Griffin P R , Calaycay J R , Ding G J , Yamin T T
Published: May 2006

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.
Cancer dormancy: isolation and characterization of dormant lymphoma cells.
Authors: Yefenof E , Picker L J , Scheuermann R H , Tucker T F , Vitetta E S , Uhr J W
Published: May 2006

"Tumor dormancy" is an operational term used to describe a prolonged quiescent state in which tumor cells are present, but tumor progression is not clinically apparent. Although clinical examples of tumor dormancy abound, little is known regarding the mechanisms underlying this state. Here we utilize an antibody-induced dormancy model of an aggressive murine B-cell lymphoma (BCL1) and show that the induction of the dormant state is accompanied by dramatic changes in tumor cell morphology and cell cycle status. These data indicate the feasibility of altering the malignant phenotype of transformed cells by specific signals originating at the cell surface, and they suggest new opportunities for therapeutic intervention in cancer.
Three inhibitors of type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication.
Authors: Li C J , Zhang L J , Dezube B J , Crumpacker C S , Pardee A B
Published: May 2006

Transcription of type 1 human immunodeficiency virus (HIV-1) provirus is governed by the viral long terminal repeat (LTR). Drugs can block HIV-1 replication by inhibiting activity of its LTR. We report that topotecan, beta-lapachone, and curcumin are potent and selective inhibitors of HIV-1 LTR-directed gene expression, at concentrations that have minor effects on cells. At these concentrations, each drug inhibited p24 antigen production in cells either acutely or chronically infected with HIV-1. Their target is transcriptional function of the LTR.
Hepatitis C virus RNA in southern African blacks with hepatocellular carcinoma.
Authors: Bukh J , Miller R H , Kew M C , Purcell R H
Published: May 2006

We used a sensitive and specific cDNA polymerase chain reaction assay to detect hepatitis C virus (HCV) RNA in serum samples from 128 unselected southern African Blacks with hepatocellular carcinoma (HCC) and found HCV RNA in 26 (20.3%) of these patients. Antibodies to HCV (anti-HCV) were detected in 59 patients (46.1%) with a first-generation ELISA test, and only 19 of these patients were HCV RNA-positive. Anti-HCV was detected in 25 patients (19.5%) with a second-generation ELISA test, and 17 of these patients were HCV RNA-positive. Among the second-generation ELISA- and HCV RNA-positive patients, 14 were anti-HCV positive, 2 were indeterminate, and 1 was anti-HCV negative in a second-generation recombinant immunoblot assay, whereas all patients who were second-generation ELISA positive, but HCV RNA negative, were anti-HCV negative in this immunoblot assay. A total of 5 patients were negative in both ELISA tests but were HCV RNA positive. Seventy-one patients (55.5%) had evidence of a current HBV infection, 50 (39.1%) had evidence of a previous infection, and 7 (5.5%) had no evidence of a current or previous HBV infection. The prevalence of current HBV infection was significantly lower in patients who were positive for HCV RNA than in those who were negative (P = 0.001). This difference was not dependent on sex, age, or geographical location of the patients. The mean age of HCC patients positive for HCV RNA (52.3 years) was significantly higher (P < 0.001) than that of negative patients (40.3 years), and the difference was not dependent on HBV status or geographical location. Patients positive for HCV RNA were more likely to be urban than were negative patients. Thus, a significant number of southern African Blacks with HCC had a current HCV infection but not a current HBV infection, further suggesting that infection with HCV plays a role, albeit minor, in the development of HCC in this population.
Evolution of maize inferred from sequence diversity of an Adh2 gene segment from archaeological specimens.
Authors: Goloubinoff P , Pääbo S , Wilson A C
Published: May 2006

A segment of the nuclear gene encoding alcohol dehydrogenase 2 (Adh2) was amplified and sequenced from extracts of archaeological maize specimens up to 4700 years old and from contemporary samples. Sequence diversity in ancient maize equals that of contemporary maize. Some ancient Adh2 alleles are identical or closely related to contemporary alleles. The data suggest that the gene pool of maize is millions of years old and that domestic races of maize stem from several wild ancestral populations.
Interference with major histocompatibility complex class II-restricted antigen presentation in the brain by herpes simplex virus type 1: a possible mechanism of evasion of the immune response.
Authors: Lewandowski G A , Lo D , Bloom F E
Published: May 2006

Host survival of herpes simplex virus type 1 (HSV-1) infection depends on the establishment of latent infections in both peripheral and central nervous systems. Strains of HSV-1 that are successful in escaping the immune response produce a lethal infection. We now report a possible mechanism of immune response evasion used by HSV-1. After intraocular inoculation of mice, HSV-1 strain F established a latent infection in the brain, whereas strain KOS did not. The immune response to HSV-1 infection (strains KOS and F) in the brain was characterized by induction of major histocompatibility complex class II expression and recruitment of CD4+ and CD8+ cells to highly restricted sites of intracerebral viral infection. Major histocompatibility complex class II antigen expression was primarily intracellular in strain KOS infection centers and at the cell surface in strain F infection centers. We propose that major histocompatibility complex class II-restricted viral-antigen presentation to T cells is interrupted during strain KOS infections, thereby allowing KOS infection to evade T-cell-mediated events that would normally protect the host from a lethal infection. Immunocompromised mice (athymic or irradiate mice) could not survive strain F infections; however, latent F infections were established in irradiated mice reconstituted with naive lymph node and spleen cells. These data suggest that class II-restricted presentation of viral antigens is required for the control of HSV-1 infections in the nervous system.
Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity.
Authors: Hunt J E , Simpson R J , Krilis S A
Published: May 2006

beta 2-Glycoprotein I (beta 2-GPI), a phospholipid-binding plasma protein, is an absolute requirement (cofactor) for the binding of autoimmune-type anti-cardiolipin (aCL) antibodies to cardiolipin (CL). The nature of this cofactor activity and the specific regions of the molecule involved have not yet been determined. We have identified a preparation of beta 2-GPI that lacks aCL antibody cofactor activity. Analysis of the structural differences between the active and inactive forms enabled identification of the region of beta 2-GPI critically important for aCL cofactor activity. The active form of beta 2-GPI bound CL and displayed cofactor activity down to 1 microgram/ml. The inactive form failed to bind CL and possessed no cofactor activity even at concentrations up to 94 micrograms/ml, indicating that the ability of beta 2-GPI to bind lipids is an absolute requirement for aCL cofactor activity. Both forms possessed identical N-terminal sequences and were recognized as essentially immunoreactively identical by polyclonal antisera to beta 2-GPI. However, the inactive form has undergone proteolytic cleavage and exists primarily as a "clipped" molecule, the polypeptide chain being cleaved between Lys-317 and Thr-318 (a potential thrombin cleavage site), with the two cleaved segments linked as a disulfide-bonded complex. This indicates that the C-terminal region is critically important for beta 2-GPI to bind lipid and for aCL cofactor activity. The clipped form of beta 2-GPI would not be suitable for use as aCL cofactor and its use may have led some investigators to conclude incorrectly that beta 2-GPI does not interact with aCL antibodies.
Chicken neuronal acetylcholine receptor alpha 2-subunit gene exhibits neuron-specific expression in the brain and spinal cord of transgenic mice.
Authors: Daubas P , Salmon A M , Zoli M , Geoffroy B , Devillers-Thiéry A , Bessis A , Médevielle F , Changeux J P
Published: May 2006

Transgenic mice carrying the complete structural gene of the alpha 2 subunit of the chicken neuronal nicotinic acetylcholine receptor (nAChR) and 7 kilobase pairs (kbp) of 5' upstream and 3 kbp of 3' downstream sequences have been generated. The transgene was stably integrated in transgenic lines and transmitted to their progeny. Avian transgene expression was predominant in the central nervous system as detected by specific alpha 2-subunit cDNA amplification. Moreover, in at least two independent mouse lines, its expression appeared to be neuron-specific and reproducibly restricted to subregions in the brain and spinal cord, as revealed by in situ hybridization histochemistry. Most cranial motor nuclei were positive, and several of the alpha 2-subunit transgene-expressing structures corresponded to cholinergic areas in rodents. This study reveals that regulatory mechanisms giving rise to neuronal-specific gene expression have been conserved at least in part between birds and mammals.
A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.
Authors: Strom M S , Nunn D N , Lory S
Published: May 2006

Precursors of the type IV pilins of a number of bacterial pathogens, as well as related proteins involved in extracellular protein export and DNA uptake, are synthesized with short basic leader sequences. Maturation of these proteins involves two consecutive posttranslational modifications. The leader sequence is first proteolytically removed by specialized endopeptidases, of which the prototype is encoded by the pilD gene of Pseudomonas aeruginosa. Subsequently, the amino termini of these proteins are methylated. Here we demonstrate that PilD, in addition to cleaving the amino-terminal leader sequences of prepilin, also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin, using S-adenosyl-L-methionine as a methyl donor. Thus, to our knowledge, PilD is the first characterized bacterial N-methyltransferase. Complete inhibition of N-methylation, but not peptide cleavage, by structural analogues of S-adenosyl-L-methionine suggests that PilD is a bifunctional enzyme with proteolytic and methylation activities carried out within two distinct active sites.
cDNA and protein sequence of polymorphic macaque albumins that differ in bilirubin binding.
Authors: Watkins S , Sakamoto Y , Madison J , Davis E , Smith D G , Dwulet J , Putnam F W
Published: May 2006

The rhesus monkey, Macaca mulatta, exhibits a geographically restricted polymorphism of serum albumins Mac A and Mac B that is recognized by electrophoresis and is associated with a difference in bilirubin-binding parameters. To identify the basis of the polymorphism, the cDNA and protein sequences of serum albumin from M. mulatta were determined. Screening of a lambda gt11 rhesus liver cDNA library yielded a 1988-bp cDNA sequence that encodes the complete amino acid sequence of mature albumin, the entire propeptide, and part of the prepropeptide. Isoelectric focusing and amino-terminal protein sequencing of CNBr fragments of albumin from A/A and B/B homozygotes were performed, and the structural difference was localized to a CNBr fragment (MCB3) spanning residues 124-264. Sequence analysis of lysyl endopeptidase peptides of MCB3 established that Mac A albumin has a glutamine residue at position 188 while the Mac B albumin has a glutamic residue at the same position. PCR amplification, subcloning, and DNA sequence analysis of clones from A/A and B/B homozygotes confirmed the protein sequence data and the codon difference of CAA versus GAA, respectively. Comparison of macaque and human serum albumin shows a 93.5% identity at the amino acid level. In human serum albumin, Glu188 is located close to the IIA binding pocket for ligands, probably including bilirubin. Derivatives of coumarin compete more efficiently with bilirubin for binding sites on the Mac A albumin than on the Mac B albumin. In regions where coumarin-containing plants are important food resources, Mac B albumin may confer a selective advantage because bilirubin is less readily displaced from it.