bluuebunny/crossref_metadata_2025 huggingface dataset:
A functional isoform of the human granulocyte/macrophage colony-stimulating factor receptor has an unusual cytoplasmic domain.
Authors: Crosier K E , Wong G G , Mathey-Prevot B , Nathan D G , Sieff C A
Published: May 2006

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) transduces a signal that results in the proliferation, differentiation, and functional activation of hematopoietic cells. This study sought to determine whether functional isoforms of the receptor exist that may be important in generating this diversity of cellular response. We have isolated a cDNA encoding an isoform of the low-affinity human GMR that is a product of alternative splicing of the GMR gene and results in a predicted 410-amino acid protein with a cytoplasmic domain that is rich in serine residues, a feature of regions critical in signal transduction for other receptors of the hematopoietin receptor superfamily. This receptor bound ligand and was functionally active when introduced into a murine factor-dependent cell line; mRNA transcripts representative of this isoform were coexpressed with those for a previously isolated 400-amino acid isoform of the GMR in normal hematopoietic and leukemic cells. In view of the recent isolation of a cDNA, designated GM-CSF R beta, that confers high-affinity binding of GM-CSF in cotransfection experiments with the low-affinity receptor, we suggest that the previously isolated low-affinity receptor be designated GM-CSF R alpha 1 and the one described in this report be designated GM-CSF R alpha 2.
Transformation-dependent activation of urokinase-type plasminogen activator by a plasmin-independent mechanism: involvement of cell surface membranes.
Authors: Berkenpas M B , Quigley J P
Published: May 2006

Transformed cells produce elevated levels of the urokinase-type plasminogen activator (u-PA), which has been linked with the invasive or migratory phenotype of these cells. The u-PA is secreted and normally maintained in the inactive, single-chain form (scu-PA) and it has been assumed that natural activation occurs via a plasmin-mediated cleavage converting scu-PA to the active, two-chain form (tcu-PA). We now demonstrate that secreted scu-PA in Rous sarcoma virus-transformed chicken embryo fibroblast (RSVCEF) cultures is activated by an endogenous, plasmin-independent mechanism. Normal CEFs and CEFs infected with a temperature-sensitive RSV mutant and incubated at the nonpermissive temperature do not activate scu-PA. Conditioned medium harvested from plasmin-free cultures of RSVCEFs contains active tcu-PA as determined by two independent methods. The scu-PA is progressively converted with time in culture and requires the presence of intact cells or a plasma membrane-enriched fraction. When added to RSVCEF cultures, a synthetic peptide corresponding to residues 20-41 of the growth factor domain of chicken u-PA blocks the conversion to tcu-PA, and scu-PA accumulates in the cultures. These results suggest that scu-PA is secreted by cells, becomes bound to a u-PA receptor, and is proteolytically converted to active tcu-PA by a catalytic mechanism on the surface of RSV-transformed fibroblasts.
Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma.
Authors: Metzger A K , Sheffield V C , Duyk G , Daneshvar L , Edwards M S , Cogen P H
Published: May 2006

We detected a germ-line mutation of the p53 gene in a patient with a malignant ependymoma of the posterior fossa. This mutation, which was found at codon 242, resulted in an amino acid substitution in a highly conserved site of exon 7 of the p53 gene; the same mutation was found in both the germ-line and the tumor tissue. This is the most common region of previously described somatic p53 mutations in tumor specimens and of the germ-line p53 mutations in patients with the Li-Fraumeni cancer syndrome. Evaluation of the patient's family revealed several direct maternal and paternal relatives who had died at a young age from different types of cancer. The association of a germ-line p53 mutation with an intracranial malignancy and a strong family history of cancer suggests that p53 gene mutations predispose a person to malignancy and, like retinoblastoma mutations, may be inherited.
Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.
Authors: Harrison S A , Buxton J M , Czech M P
Published: May 2006

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.
Cloning and expression of a rat cardiac delayed rectifier potassium channel.
Authors: Paulmichl M , Nasmith P , Hellmiss R , Reed K , Boyle W A , Nerbonne J M , Peralta E G , Clapham D E
Published: May 2006

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.
Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription.
Authors: Surratt C K , Milan S C , Chamberlin M J
Published: May 2006

Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.
Optimized strategies for sequence-tagged-site selection in genome mapping.
Authors: Palazzolo M J , Sawyer S A , Martin C H , Smoller D A , Hartl D L
Published: May 2006

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.
Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.
Authors: Berthold H K , Hachey D L , Reeds P J , Thomas O P , Hoeksema S , Klein P D
Published: May 2006

The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.
Neurotrophic activity of platelet-derived growth factor (PDGF): Rat neuronal cells possess functional PDGF beta-type receptors and respond to PDGF.
Authors: Smits A , Kato M , Westermark B , Nistér M , Heldin C H , Funa K
Published: May 2006

The different isoforms of PDGF bind with different affinities to two different receptor types. Previously, these receptors have been demonstrated mainly on mesenchymal and glial cells. We show here that PDGF beta-type receptors are present also on rat brain neurons in vivo as well as in vitro. Immunohistochemical analysis of a sequentially sectioned brain of a newborn rat revealed the presence of PDGF beta receptors in neurons throughout the central nervous system. These beta-receptor-bearing cells were shown to contain neurofilaments by double immunofluorescence staining. Furthermore, the PDGF-BB isoform bound to cells in a neuron-enriched culture from newborn rat brains and induced c-Fos protein in these cells. Continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival. These results indicate a neurotrophic effect of PDGF-BB.
Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.
Authors: Raines M A , Liu L , Quan S G , Joe V , DiPersio J F , Golde D W
Published: May 2006

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in hematopoiesis and host defense via interaction with specific cell-surface receptors in target tissues. We identified a truncated, soluble form of the low-affinity GM-CSF receptor (GMR) in chorio-carcinoma cells. Low-affinity GMR cDNAs encoding both the membrane-bound and soluble receptors were obtained by PCR using primers corresponding to the published sequence. Clones encoding the soluble receptor were identical in sequence to the membrane-bound form but contained a 97-nucleotide internal deletion. The amino acid sequence of this deleted cDNA predicts a protein that lacks the 84 C-terminal amino acids of the membrane-bound receptor, including the transmembrane and cytoplasmic domains, and contains 16 different amino acids at its C terminus. Expression of the soluble GMR cDNA in murine psi-AM cells as well as GM-CSF-dependent myeloid 32Dc13 cells produced a secreted protein that retained its capacity to bind GM-CSF in solution. RNase protection analysis indicates that this variant cDNA is derived from a naturally occurring mRNA. Soluble receptors have been identified for several other hematopoietin receptors and may be a general feature of this class. The striking similarity between the soluble form of the GMR and other hematopoietin receptors suggests that soluble binding proteins may play an important role in regulating the broad spectrum of biological responses mediated by these cytokines.
Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.
Authors: Postel E H , Flint S J , Kessler D J , Hogan M E
Published: May 2006

A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.
A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.
Authors: Gaspar M L , Meo T , Bourgarel P , Guenet J L , Tosi M
Published: May 2006

Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation.
Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line.
Authors: Homaidan F R , Sharp G W , Nowak L M
Published: May 2006

Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects.
Adenovirus enhancement of transferrin-polylysine-mediated gene delivery.
Authors: Curiel D T , Agarwal S , Wagner E , Cotten M
Published: May 2006

Gene transfer may be accomplished by the receptor-mediated endocytosis pathway using transferrin-polylysine conjugates. For some target cells, however, gene transfer by this vector is extremely limited, despite the presence of the appropriate surface receptors, a phenomenon attributed to lysosomal degradation of endosome-internalized conjugate-DNA complexes. To enhance DNA escape from the cell vesicle system and thus augment gene transfer by this route, we have used the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism. Adenoviral infection augmented levels of gene transfer by transferrin-polylysine conjugates in a dose-dependent manner: levels of gene transfer of greater than 2000-fold above baseline were achieved. Use of the adenovirus in this context allowed enhanced levels of gene transfer in a variety of target cells, including cell lines otherwise refractory to gene transfer by transferrin-polylysine conjugates. This augmentation was based on adenoviral-mediated vesicle disruption, a process independent of viral gene expression. Thus, the development of specific mechanisms to effect release from the endosome in combination with gene transfer by the receptor-mediated endocytosis pathway will increase the utility of this delivery system by allowing high levels of gene expression in target cells.
Identification of nuclear proteins that specifically bind to RNAs containing 5' splice sites.
Authors: Stolow D T , Berget S M
Published: May 2006

Two polypeptides of 26 and 37 kDa (designated SPP-1 and SPP-2) were identified in in vitro splicing extracts by UV crosslinking to splicing precursor RNAs. Crosslinking of both polypeptides required a functional 5' splice site but was not dependent on sequences at the 3' end of the intron. Centrifugation of extract separated the two polypeptides from major U small nuclear ribonucleoproteins (snRNPs), including U1 snRNPs. Both polypeptides crosslinked to precursor RNAs containing 5' splice sites in the absence of U1 RNA. Complexes containing both polypeptides also contained U1 snRNPs, suggesting that SPP-1 and SPP-2 are a part of the functional spliceosome. We propose that SPP-1 and SPP-2 are factors that participate in the recognition of 5' splice sites.
Structure-function studies of human aromatase by site-directed mutagenesis: kinetic properties of mutants Pro-308----Phe, Tyr-361----Phe, Tyr-361----Leu, and Phe-406----Arg.
Authors: Zhou D J , Pompon D , Chen S A
Published: May 2006

Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [1 beta,2 beta-3H]androstenedione, [19-14C]androstenedione, and [1 beta,2 beta-3H]testosterone as substrates. The mutant Phe-406----Arg was completely inactive. Since there were only small changes in the Km and Vmax values for all substrates for mutants Tyr-361----Phe and Tyr-361----Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308----Phe had altered catalytic properties; the Km values for androstenedione, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.
Cloning and expression of a cDNA encoding human sterol carrier protein 2.
Authors: Yamamoto R , Kallen C B , Babalola G O , Rennert H , Billheimer J T , Strauss J F
Published: May 2006

We report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP2). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP2. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A)+ RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP2 gene in the human genome or that the SCP2 gene is very large. Coexpression of the SCP2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP2 plays a role in regulating steroidogenesis, among other possible functions.
X-ray scattering indicates that the leucine zipper is a coiled coil.
Authors: Rasmussen R , Benvegnu D , O'Shea E K , Kim P S , Alber T
Published: May 2006

Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper. We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4. This peptide is known to form a dimer of parallel helices in solution. X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils. The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils. There is no diffraction maximum near 5.4 A, the characteristic pitch of straight helices. Our results provide direct evidence that the leucine zipper of GCN4 is a coiled coil.
Molecular dynamics simulation of the hydration shell of a B-DNA decamer reveals two main types of minor-groove hydration depending on groove width.
Authors: Chuprina V P , Heinemann U , Nurislamov A A , Zielenkiewicz P , Dickerson R E , Saenger W
Published: May 2006

The conformation of the self-complementary B-DNA decamer C-C-A-A-C-G-T-T-G-G is known from a high-resolution x-ray crystal structure analysis. Molecular dynamics simulation of the hydration shell of the decamer has revealed two main types of minor-groove hydration, depending on groove width. The narrow part of the minor groove has a spine of hydration analogous to that described for the A + T-rich center of the minor groove in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G [Drew, H. R. & Dickerson, R. E. (1981) J. Mol. Biol. 151, 535-556], the first hydration layer of which contains one water molecular per base pair. In contrast, in the wide part of the minor groove, each base is hydrated individually, water molecules lying predominantly in the base plane. In intermediate-width regions, preferred water-molecule sites are shifted away from the base plane in a 3'-to-5' direction. This shift becomes more pronounced as the minor groove narrows, until the two water molecules lie approximately midway between base pairs. If the minor groove is narrowed still further, it accommodates only one water molecule, and the hydration transforms to the well-known water spine. The observed pattern agrees with available crystallographic data and with our earlier calculations. The results confirm the assumption that preferred positions of water oxygens in the minor groove depend predominantly on groove width rather than on base sequence. However, the location of water hydrogens, and the network of hydrogen bonding, can depend on base sequence. We suggest a simple explanation of water-spine formation in the narrow minor groove of a random DNA sequence. The spine of hydration may be a property of the minor groove of overwound variants of B-DNA, the C and D forms, for which the middle part of the decamer C-C-A-A-C-G-T-T-G-G can serve as a model.
Inhibition of sleep in rats by inorganic selenium compounds, inhibitors of prostaglandin D synthase.
Authors: Matsumura H , Takahata R , Hayaishi O
Published: May 2006

Prostaglandin (PG) D2 has been postulated to be an endogenous sleep-promoting factor in rats, and SeCl4 and Na2SeO3 recently have been shown to inhibit the PGD synthase (prostaglandin-H2 D-isomerase, EC 5.3.99.2) activity of rat brain. The effect of these selenium compounds on sleep-wake activities was examined in freely moving rats along with their effects on brain temperature, food and water intake, and behavior. Test substances were administered for 6 hr into the third ventricle of rats, using a microdialysis technique. SeCl4, time- and dose-dependently, inhibited sleep at perfusion rates of 60 pmol/0.2 microliter per min and higher, and the inhibition was almost complete at rates greater than 200 pmol/0.2 microliter per min. The effect was reversible and was followed by a rebound. Na2SeO3 exhibited similar effects, but Na2SO3 did not show any effect on sleep. Simultaneous administration of dithiothreitol eliminated the sleep-inhibiting effects of these selenium compounds. These findings indicate that the decrease in sleep is due to inhibition of the PGD synthase activity in the brain by SeCl4 as well as Na2SeO3. During the inhibition of sleep, the rats in general showed an activation of behavior with moderate elevation of brain temperature and a detectable increase in food and water intake, suggesting that the sleep-inhibited state of the rats was similar to the physiological state of wakefulness and that the inhibitory effect was not due to the general toxicity of selenium.
Saccharide orientation at the cell surface affects glycolipid receptor function.
Authors: Strömberg N , Nyholm P G , Pascher I , Normark S
Published: May 2006

Three allelic variants of P-pilus-associated G-adhesins (lectins) with different cell-binding properties were recently described. Here we have analyzed Escherichia coli HB101 strains expressing the recombinant G-adhesin variants for their ability to agglutinate erythrocytes from various species as this relates to the glycosphingolipid (GSL) composition in the erythrocyte membranes. All three variants exhibit similar specificities for the globo-series GSLs affixed to artificial surfaces. However, only the PapGJ96 adhesin induces agglutination of erythrocytes having globotriaosylceramide (GbO3) [Gal(alpha 1-4)LacCer] as the major GSL. Furthermore, only PapGAD110 induces strong agglutination of erythrocytes having globotetraosylceramide (GbO4) [GalNAc(beta 1-3)Gal(alpha 1-4)LacCer] as the major GSL, while PrsGJ96 alone agglutinates those containing globopentaosylceramide (GbO5) [GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)LacCer]. Molecular modeling of these globo-GSLs demonstrates different saccharide orientations to the membrane surface for these isoreceptors. We suggest that the differential binding of the three G-adhesin variants results from differences in epitope presentation at the membrane among these globo-GSLs.
Multiple murine alpha 1-protease inhibitor genes show unusual evolutionary divergence.
Authors: Borriello F , Krauter K S
Published: May 2006

The murine alpha 1-protease inhibitors (alpha 1-PI) are encoded by a small gene family on chromosome 12. Studies of alpha 1-PI and other serine protease inhibitor genes have revealed an unusually high rate of mutation of the reactive centers of the inhibitors. Using a modification of the PCR technique, we have previously identified five distinct alpha 1 PI reactive site sequences present in the genome of C57BL/6 mice. In this report, we use cDNA cloning techniques to demonstrate that all five genes are expressed in the adult mouse liver. DNA sequence analysis shows that three of the five mRNAs expressed have a substitution for methionine-353, which is essential for normal activity of the homologous human protein, alpha 1-antitrypsin (alpha 1-AT). Comparison of the DNA sequences of the five cDNAs indicates a higher degree of polymorphism in the carboxyl-terminal half of the protein and an extraordinarily replacement/silent ratio of nucleotide changes in a narrow region surrounding the reactive site. The clustering of polymorphisms near the reactive site combined with the high replacement/silent ratio suggest an evolutionary mechanism that apparently selects for functional diversity of the alpha 1-PI genes. Finally, modeling of the three-dimensional positions of the alpha 1-PI polymorphic residues into the homologous positions of the crystallographic structure of ovalbumin, a member of the alpha 1-PI supergene family, predicts that many of these amino acids are on the surfaces, which are likely to interact with the protease targets.
Nonresponsiveness to an immunodominant Epstein-Barr virus-encoded cytotoxic T-lymphocyte epitope in nuclear antigen 3A: implications for vaccine strategies.
Authors: Schmidt C , Burrows S R , Sculley T B , Moss D J , Misko I S
Published: May 2006

An immunodominant Epstein-Barr virus (EBV)-encoded cytotoxic T lymphocyte (CTL) epitope has been mapped to the EBV nuclear antigen 3A. The epitope, represented by the peptide sequence AWNAGFLRGRAYGLD (hereafter termed AWNA), is restricted through the HLA-B8 allele and is expressed by type A but not type B-infected transformants. Herein, we show that EBV-specific memory CTLs from an HLA-B8+ healthy virus carrier, JS, did not respond in vitro to AWNA, even though that individual's endogenously infected transformants processed and presented the natural equivalent of this peptide to AWNA-specific CTLs from another B8+ individual. Instead, an epitope, represented by the peptide sequence QLSDTPLIPLTIFVGENTGV, was the dominant EBV-specific CTL epitope in donor JS. This epitope mapped to EBV nuclear antigen 2A, was restricted by an HLA-A2 subtype, and specifically associated with type A strains of EBV. No AWNA-specific CTL precursors were detected by limiting dilution analysis of peripheral blood mononuclear cells from donor JS whereas the precursor frequency of AWNA-specific CTLs from a responder donor, LC, was estimated at 1:4500. The presentation in vivo of an immunogenic epitope-HLA antigen complex is clearly insufficient to guarantee an effective memory CTL response to that foreign epitope. Thus, vaccination strategies based on peptides inducing CTL responses may need to take into account not only the polymorphism of HLA antigens but also possible allelic variation in the repertoires of T-cell receptors.
Gene targeting in Chinese hamster ovary cells is conservative.
Authors: Pennington S L , Wilson J H
Published: May 2006

Two fundamentally different pathways for homologous recombination have been identified in mammalian cells. For most chromosomal recombination events, two copies of a homologous sequence recombine to yield two copies in the products; such events are said to be conservative because the number of copies is preserved. By contrast, virtually all extrachromosomal recombination events are nonconservative; two copies recombine to give a product containing a single intact copy (the other copy is destroyed in the mechanism). Since gene targeting involves an introduced (extrachromosomal) plasmid and a chromosomal target, it was not clear which pathway would apply. We used a marked vector to determine whether targeted integrants were products of recombination events that involved two copies (the conservative pathway) or three copies (the nonconservative pathway) of the homologous sequence. Among 51 gene targeting events, we identified 17 homologous integrants and analyzed their structures. All match the predictions for a conservative pathway. We conclude that the principal pathway for gene targeting in mammalian cells is conservative.
DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity.
Authors: McCarty J S , Walker G C
Published: May 2006

DnaK, the sole Escherichia coli member of the highly conserved 70-kDa heat shock protein (HSP70) family of proteins, autophosphorylates when incubated with ATP in vitro. We show that threonine-199 is the amino acid that becomes phosphorylated and we demonstrate that threonine-199 is critical for the ATPase activity of DnaK. We also report that both the ATPase and autophosphorylating activities of DnaK increase very strongly over the range of temperatures that is physiologically relevant for E. coli growth. The temperature dependence of either or both of these activities could be of significance with respect to the postulated role of DnaK as a molecular chaperone in helping cells ameliorate the deleterious consequences of elevated temperature. Furthermore, we postulate that DnaK plays a key role in regulation of the heat shock response by serving as a cellular thermometer that directly senses the environmental temperature.
Identification of a continuous and cross-reacting epitope for Plasmodium falciparum transmission-blocking immunity.
Authors: Wizel B , Kumar N
Published: May 2006

Identification of continuous epitopes in the target antigens of Plasmodium falciparum transmission-blocking antibodies is likely to facilitate the production of a subunit peptide vaccine. Two such epitopes shared among several sexual-stage antigens were identified with murine monoclonal antibodies. An epitope recognized by four monoclonal antibodies capable of blocking infectivity of gametocytes in the mosquitoes is shared among three antigens (230, 48/45 doublet, and 27 kDa). These antigens are synthesized at different times during the development and maturation of gametocytes, and the blocking epitope appears conserved among parasites from diverse geographical locations. Immune response against such a unique epitope (continuous, cross-reacting, and conserved) is likely to be boosted by natural infection. The 27-kDa protein is reported here as a target of malaria transmission-blocking monoclonal antibodies, and the cross-reacting epitope represents an attractive candidate for a transmission-blocking vaccine.
Sequence of general transcription factor TFIIB and relationships to other initiation factors.
Authors: Malik S , Hisatake K , Sumimoto H , Horikoshi M , Roeder R G
Published: May 2006

Transcription factor TFIIB is a ubiquitous factor required for transcription initiation by RNA polymerase II. Previous studies have suggested that TFIIB serves as a bridge between the "TATA"-binding factor (TFIID) and RNA polymerase II during preinitiation complex assembly and, more recently, that TFIIB can be a target of acidic activators. We have purified TFIIB to homogeneity, shown that activity resides in a 33-kDa polypeptide, and obtained cDNAs encoding functional TFIIB. TFIIB contains a region with amino acid sequence similarity to a highly conserved region of prokaryotic sigma factors. This is consistent with analogous functions for these factors in promoter recognition by RNA polymerases and with similar findings for TFIID, TFIIE, and TFIIF/RAP30. Like TFIID, TFIIB contains both a large imperfect repeat that could contribute an element of symmetry to the folded protein and clusters of basic residues that could interact with acidic activator domains. These findings argue for a common origin of TFIIB, TFIID, and other general transcription factors and for the evolutionary segregation of complementary functions.
Characterization and mapping of human genes encoding zinc finger proteins.
Authors: Bray P , Lichter P , Thiesen H J , Ward D C , Dawid I B
Published: May 2006

The zinc finger motif, exemplified by a segment of the Drosophila gap gene Krüppel, is a nucleic acid-binding domain present in many transcription factors. To investigate the gene family encoding this motif in the human genome, a placental genomic library was screened at moderate stringency with a degenerate oligodeoxynucleotide probe designed to hybridize to the His/Cys (H/C) link region between adjoining zinc fingers. Over 200 phage clones were obtained and are being sorted into groups by partial sequencing, cross-hybridization with oligodeoxynucleotide probes, and PCR amplification. Further, the genomic clones were cross-hybridized with a set of 30 zinc finger-encoding cDNAs (Kox1-Kox30) isolated from a human T-cell cDNA library. Four cDNAs (Kox4, Kox7, Kox12, and Kox15) were identified that match one or more genomic clones; these matches were confirmed by nucleotide sequence analysis. One or more clones from each locus were mapped onto human metaphase chromosomes by chromosomal in situ suppression hybridization with fluorescent probe detection. We mapped ZNF7/Kox4 to chromosome 8qter, ZNF19/Kox12 to 16q22, ZNF22/Kox15 to 10q11, and ZNF44/Kox7 to 16p11. The results of these analyses support the conclusion that the human genome contains many, probably several hundred, zinc finger genes with consensus H/C link regions.
Quantitative assay for morphogenesis indicates the role of extracellular matrix components and G proteins.
Authors: Klebe R J , Overfelt T M , Magnuson V L , Steffensen B , Chen D L , Zardeneta G
Published: May 2006

A quantitative assay for morphogenesis is described that involves counting the organizing centers (swirling patterns) formed by many cultured fibroblasts. Organizing centers, which are found in vivo, represent one of the smallest units of morphogenesis. We show that macroscopically visible organizing centers form by the merger of smaller organizing centers. Parallel orientation of cells on plastic substrata requires cell-cell contact, but organizing centers can develop without cell-cell contact on collagen gels. On collagen gels, the orientation of collagen fibers determines the orientation of cells with respect to one another. Although organizing centers resemble fingerprints, we have shown that a stochastic process determines the spatial orientation of organizing centers. Treatment of transformed cell lines with agents that increase cAMP levels or alter the activity of guanine nucleotide binding proteins resulted in the generation of organizing centers. Cholesterol precursors involved in protein isoprenylation were found to be potent reverse-transformation agents that could alter the two-dimensional morphogenesis of cells. The simple assay described should permit the analysis of morphogenesis at the molecular and cellular levels.
PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma.
Authors: Rosenberg C L , Wong E , Petty E M , Bale A E , Tsujimoto Y , Harris N L , Arnold A
Published: May 2006

Rearrangement of the BCL1 (B-cell lymphoma 1) region on chromosome 11q13 appears to be highly characteristic of centrocytic lymphoma and also is found infrequently in other B-cell neoplasms. Rearrangement is thought to deregulate a nearby protooncogene, but transcribed sequences in the immediate vicinity of BCL1 breakpoints had not been identified. PRAD1, previously designated D11S287E, was identified on 11q13 as a chromosomal breakpoint region rearranged with the parathyroid hormone gene in a subset of parathyroid adenomas; this highly conserved putative oncogene, which encodes a novel cyclin, has been linked to BCL1 and implicated also in subsets of breast and squamous cell neoplasms with 11q13 amplification. We report pulsed-field gel electrophoresis data showing BCL1 and PRAD1 to be no more than 130 kilobases apart. PRAD1 mRNA is abundantly expressed in seven of seven centrocytic lymphomas (Kiel classification), in contrast to 13 closely related but noncentrocytic lymphomas. Three of the seven centrocytic lymphomas had detectable BCL1 DNA rearrangement. Also, two unusual cases of CLL with BCL1 rearrangement overexpressed PRAD1, in contrast to five CLL controls. Thus, PRAD1 is an excellent candidate "BCL1 oncogene." Its overexpression may be a key consequence of rearrangement of the BCL1 vicinity in B-cell neoplasms and a unifying pathogenetic feature in centrocytic lymphoma.
A neural network model of sensoritopic maps with predictive short-term memory properties.
Authors: Droulez J , Berthoz A
Published: May 2006

Coordinated orienting movements can be accurately performed without direct sensory control. Ocular saccades, for instance, have been shown to be reprogrammed after target disappearance when an intervening eye movement is electrically triggered before the saccade onset. Saccadic eye movements can also be executed toward memorized targets, even when the subject has been passively moved in darkness. Two hypotheses have been proposed to account for this goal-invariance property: either (i) the goal is reconstructed and memorized in the stable frame of reference linked to the environment ("allocentric, coordinates") or (ii) the goal is selected and memorized in the sensors-related maps ("egocentric coordinates") and is continuously updated by efferent copies of the motor commands. In this paper, we shall describe a formal neural network based on this second hypothesis. The results of the simulation show that target position can be memorized and accurately updated in a topologically ordered map, using a velocity-signal feedback. Moreover, this network has been submitted to a simple learning procedure by using the intermittent visual recurring afferent signal as the teaching signal. A similar mechanism could be involved in control of limb movement.
Early tertiary elephant-shrews from Egypt and the origin of the Macroscelidea.
Authors: Simons E L , Holroyd P A , Bown T M
Published: May 2006

Recent expeditions to the Fayum Depression, Egypt, have made possible the discovery of mandibles and a maxilla of a new genus and species of late Eocene elephant-shrew as well as initial evidence of the upper dentition of the early Oligocene taxon Metoldobotes. These fossils demonstrate that macroscelideans underwent a significant radiation in the Early Tertiary of Africa. Two new subfamilies are recognized and described. These Tertiary macroscelideans are the most primitive elephant-shrews known and indicate that previous hypotheses of a close phylogenetic relationship between macroscelideans and either lagomorphs, erinaceotans, or tree-shrews are unlikely. Rather, the dental anatomy of the Fayum macroscelideans provides evidence for a derivation of the order from within the Condylarthra.
Reverse transcriptase encoded by a retrotransposon from the trypanosomatid Crithidia fasciculata.
Authors: Gabriel A , Boeke J D
Published: May 2006

The long interspersed nuclear element (LINE)-like elements are a distinct family of eukaryotic transposons that contain a long open reading frame with limited sequence homology to retroviral reverse transcriptases. Unlike many retrotransposons, they lack long terminal repeats. The mechanism by which LINE-like elements move within the genomes of their hosts remains speculative. We have used an unusual approach to express and detect enzymatic activities associated with Crithidia retrotransposable element 1 (CRE1), a site-specific LINE-like element found in the insect trypanosomatid Crithidia fasciculata. A chimeric gene fusing the yeast retrotransposon Ty1 and the CRE1 open reading frame is constructed and then overexpressed in yeast. Fusion proteins are packaged into virus-like particles, which can be partially purified and directly analyzed for enzymatic activity. Here we demonstrate that CRE1 encodes an RNA-directed DNA polymerase. These data provide direct biochemical evidence that this widely distributed class of retrotransposons encodes reverse transcriptase and sets the stage for a detailed understanding of the mechanisms involved in LINE-like element transposition.