bluuebunny/crossref_metadata_2025 huggingface dataset:
Energetics of the structure of the four-alpha-helix bundle in proteins.
Authors: Chou K C , Maggiora G M , Némethy G , Scheraga H A
Published: May 2006

The main features of the four-alpha-helix bundle, one of the characteristic structural elements of many proteins, can be explained in terms of noncovalent interactions between the constituent helices. Conformational energy computations have been carried out on four types of four-alpha-helix bundles, each consisting of four CH3CO-(L-Ala)10-NHCH3 polypeptide chains, with various combinations of parallel and antiparallel orientations of the helices. In the bundle with the most favorable energy, all pairs of neighboring helices are oriented antiparallel--i.e., in the orientation that is favored by electrostatic interactions between the helices. In this structure, the orientation angle between neighboring helix axes is -168 degrees, within +/- 7 degrees, in close agreement with the orientation angles observed in proteins and with the value that we computed earlier for the most favorable packing of pairs of interacting alpha-helices. This orientation corresponds to a left-handed twisting of the helical bundle. The preferred handedness of this twisting arises as a result of favorable nonbonded interactions between the alpha-helices.
Codon preference is but an illusion created by the construction principle of coding sequences.
Authors: Ohno S
Published: May 2006

Modern coding sequences are in the periodicto-chaotic transition. In the case of two related sequences for lens alpha A-crystallin and small heat shock protein, the original repeating units were heptameric in length. Accordingly, base trimers that were parts of heptameric units recurred far more frequently than those that were not included. In the crystallin coding sequence, CTG trimer recurred 21 times, and TCT and TCC trimers recurred 17 times each. By contrast, CTA and TCG, although related to the above, recurred only 4 and 3 times, respectively. It is a small wonder that 10 of the 16 leucine residues were encoded by CTG, whereas none was encoded by CTA, and that 17 of the 23 serine residues were encoded either by TCT or by TCC, whereas only 1 was encoded by TCG. In the small heat shock protein coding sequence, however, AGC became parts of the two prominent heptameric recurring units. Not surprisingly, 10 of the 22 serine residues were now encoded by AGC. In conclusion, the so-called codon preference is a mere reflection of the construction principle of coding sequences and has very little to do with selection per se.
Differential ability of B cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific T-cell clones in vivo.
Authors: Scherle P A , Gerhard W
Published: May 2006

When a helper T-cell (TH) clone specific for the hemagglutinin, neuraminidase, matrix protein, or nucleoprotein of influenza strain A/PR/8/34 is adoptively transferred to athymic mice 1 day after virus infection the anti-viral antibody response of the mouse is enhanced. This response is directed predominantly to the hemagglutinin and requires associative T-cell-B-cell interactions. Delaying transfer of the TH clone has three consequences: (i) the onset of the anti-hemagglutinin antibody response is delayed; (ii) the titer of the anti-hemagglutinin response is reduced; and (iii) the titer of the antibody in the response against the internal proteins, matrix protein and nucleoprotein, is enhanced upon transfer of matrix protein- or nucleoprotein-specific, but not hemagglutinin- or neuraminidase-specific, TH clones. Thus, there is a hierarchy of help: B cells recognizing viral surface components, hemagglutinin or neuraminidase, can receive help from TH clones specific for any of the major structural viral proteins. In contrast, B cells responding to internal viral components, matrix protein or nucleoprotein, are restricted to receiving help almost exclusively from TH clones with the same protein specificity. These observations suggest that, upon B-cell surface immunoglobulin-antigen interaction and uptake of intact virus, B cells specific for viral surface proteins process and present all major structural viral antigens, enabling the B cells to interact with TH clones specific for any virion protein. B cells recognizing internal viral components, which may be accessible to interaction with B-cell immunoglobulin receptors mainly as free proteins, would present only the protein for which they are specific and, thereby, receive help only from the TH clones of the same protein specificity.
Endogenous adenosine and hemorrhagic shock: effects of caffeine administration or caffeine withdrawal.
Authors: Conlay L A , Evoniuk G , Wurtman R J
Published: May 2006

Plasma adenosine concentrations doubled when rats were subjected to 90 min of profound hemorrhagic shock. Administration of caffeine (20 mg per kg of body weight), an adenosine-receptor antagonist, attenuated the hemorrhage-induced decrease in blood pressure. In contrast, chronic caffeine consumption (0.1% in drinking water), followed by a brief period of caffeine withdrawal, amplified the hypotensive response to hemorrhage. These data suggest that endogenous adenosine participates in the hypotensive response to hemorrhage and that caffeine may protect against, and caffeine withdrawal may exacerbate, this response.
Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases.
Authors: Manetto V , Perry G , Tabaton M , Mulvihill P , Fried V A , Smith H T , Gambetti P , Autilio-Gambetti L
Published: May 2006

Several degenerative diseases of the nervous system are characterized by the presence of neuronal inclusions. Most of these inclusions are made of abnormal filaments and share epitopes with cytoskeletal proteins. One of these inclusions, the neurofibrillary tangle of Alzheimer disease, has recently been shown to contain ubiquitin, a regulatory protein thought to play a role in the degradation of abnormal proteins. We carried out light and electron microscopic immunocytochemistry with several polyclonal and monoclonal antibodies to investigate the presence of ubiquitin in neuronal inclusions of neurodegenerative diseases. Ubiquitin was present not only in paired helical filaments that form the neurofibrillary tangle of Alzheimer disease, but also in the filamentous components of the inclusion characteristic of Parkinson disease, Pick disease, and progressive supranuclear palsy. In contrast, ubiquitin was not detected in other neuronal inclusions often found in aging and in Alzheimer disease, such as Hirano bodies and granulovacuolar degeneration. Reactivity with monoclonal antibodies suggests differences in the ubiquitin-acceptor proteins present in the inclusions studied. It is concluded that ubiquitin is selectively present in neuronal inclusions of degenerative diseases.
Background light and the contrast gain of primate P and M retinal ganglion cells.
Authors: Purpura K , Kaplan E , Shapley R M
Published: May 2006

Retinal ganglion cells projecting to the monkey lateral geniculate nucleus fall into two classes: those projecting to the magnocellular layers of the nucleus (M cells) have a higher contrast gain to luminance patterns at photopic levels of retinal illumination than those projecting to the parvocellular layers (P cells). We report here that this difference in luminance contrast gain between M and P cells is maintained at low levels of mean retinal illumination. In fact, our results suggest that in the mesopic and scotopic ranges of mean illumination, the M-cell/magnocellular pathway is the predominant conveyor of information about spatial contrast to the visual cortex.
Antiviral agents targeted to interact with viral capsid proteins and a possible application to human immunodeficiency virus.
Authors: Rossmann M G
Published: May 2006

The tertiary structure of most icosahedral viral capsid proteins consists of an eight-stranded antiparallel beta-barrel with a hydrophobic interior. In a group of picornaviruses, this hydrophobic pocket can be filled by suitable organic molecules, which thereby stop viral uncoating after attachment and penetration into the host cell. The antiviral activity of these agents is probably due to increased rigidity of the capsid protein, which inhibits disassembly. The hydrophobic pocket may be an essential functional component of the protein and, therefore, may have been conserved in the evolution of many viruses from a common precursor. Since eight-stranded anti-parallel beta-barrels, with a topology as in viral capsid proteins, are not generally found for other proteins involved in cell metabolism, this class of antiviral agents is likely to be more virus-specific and less cytotoxic. Furthermore, the greatest conservation of viral capsid proteins occurs within this pocket, whereas the least conserved part is the antigenic exterior. Thus, compounds that bind to such a pocket are likely to be effective against a broader group of serologically distinct viruses. Discovery of antiviral agents of this type will, therefore, depend on designing compounds that can enter and fit snugly into the hydrophobic pocket of a particular viral capsid protein. The major capsid protein, p24, of human immunodeficiency virus would be a likely suitable target.
DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor.
Authors: Koudelka G B , Harbury P , Harrison S C , Ptashne M
Published: May 2006

The affinity of the Escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressor-operator complex. Central sequence effects on affinity for repressor are independent of the identity of adjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.
Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.
Authors: Clarke S , Vogel J P , Deschenes R J , Stock J
Published: May 2006

The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions.
Pentose pathway in human liver.
Authors: Magnusson I , Chandramouli V , Schumann W C , Kumaran K , Wahren J , Landau B R
Published: May 2006

[1-14C]Ribose and [2-14C]glucose were given to normal subjects along with glucose loads (1 g per kg of body weight) after administration of diflunisal and acetaminophen, drugs that are excreted in urine as glucuronides. Distributions of 14C were determined in the carbons of the excreted glucuronides and in the glucose from blood samples drawn from hepatic veins before and after glucagon administration. Eighty percent or more of the 14C from [1-14C]ribose incorporated into the glucuronic acid moiety of the glucuronides was in carbons 1 and 3, with less than 8% in carbon 2. In glucuronic acid from glucuronide excreted when [2-14C]glucose was given, 3.5-8.1% of the 14C was in carbon 1, 2.5-4.3% in carbon 3, and more than 70% in carbon 2. These distributions are in accord with the glucuronides sampling the glucose unit of the glucose 6-phosphate pool that is a component of the pentose pathway and is intermediate in glycogen formation. It is concluded that the glucuronic acid conjugates of the drugs can serve as a noninvasive means of sampling hepatic glucose 6-phosphate. In human liver, as in animal liver, the classical pentose pathway functions, not the L-type pathway, and only a small percentage of the glucose is metabolized via the pathway.
Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.
Authors: Lake J A , de la Cruz V F , Ferreira P C , Morel C , Simpson L
Published: May 2006

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.
Magnetic fields and orientation in homing pigeons: experiments of the late W. T. Keeton.
Authors: Moore B R
Published: May 2006

The late W. T. Keeton conducted 35 experiments examining the effects of bar magnets upon the navigation of experienced homing pigeons on overcast days. Six statistics summarize the consistency and accuracy of the birds' initial orientation and the speed and success of their subsequent homing. Magnets had no significant overall effect upon these measures.
Retinal afferent arborization patterns, dendritic field orientations, and the segregation of function in the lateral geniculate nucleus of the monkey.
Authors: Michael C R
Published: May 2006

Optic tract fibers and cell bodies in the lateral geniculate nucleus of the monkey were studied intracellularly with micropipette electrodes containing the marker enzyme horseradish peroxidase. Single optic-tract fibers always projected to only one of the six geniculate layers. The majority of the axons innervating the four parvocellular laminae were red/green opponent color units; their terminations formed cylindrical columns that were perpendicular to the layers. In similar fashion, the geniculate cells in the parvocellular layers were mostly red/green units with narrow, bipolar dendritic fields oriented normal to the laminar borders. The majority of the retinal axons ending in parvocellular layers 6 and 5 were on-center units; nearly all geniculate cells in these two laminae were also on-center neurons. In layers 4 and 3 most terminating optic-tract fibers, as well as the geniculate cells themselves, were off-center units. All axons projecting to the magnocellular layers were broad-band units with spherical terminal arborizations. The magnocellular geniculate neurons, which were also broad band, had extensive spherical dendritic fields that often crossed laminar borders. Thus, the terminal patterns of each class of retinogeniculate axon closely resembled the dendritic orientations of the functionally related geniculate target cells.
Antibody specific to the alpha subunit of the guanine nucleotide-binding regulatory protein Go: developmental appearance and immunocytochemical localization in brain.
Authors: Chang K J , Pugh W , Blanchard S G , McDermed J , Tam J P
Published: May 2006

A polyclonal rabbit antibody (9120) against the alpha subunit of the "other" guanine nucleotide-binding protein Go (alpha o) was raised against a synthetic alpha o peptide fragment (Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val) attached to a branched core system. Antiserum 9120, at a final dilution of 1:400, can detect alpha o in as little as 0.2 microgram of Go on immunoblots, and, at a final dilution of 1:20,000, can detect alpha o in 1 microgram of Go on immunoblots. Antiserum and affinity-purified antibody are specific to alpha o. No cross-reactivity was detected to the alpha subunits of the stimulatory or inhibitory guanine nucleotide-binding regulatory proteins or of transducin (alpha s, alpha i, or alpha T) or to the beta or gamma subunits. Immunoblots revealed a high density of alpha o in rat brain and lung membrane preparations, but other tissues (such as adipose tissue, heart, erythrocytes, and liver) have no detectable alpha o. Developmental studies showed that alpha o in rat brain was low before birth, increased after birth, and reached the full adult level at 4 weeks of age. In contrast, ADP-ribosylation of 40-kDa proteins increased for up to 1 week and then decreased. Immunocytochemistry revealed that alpha o was localized to somatic and synaptic membranes in rat brain, whereas little or no alpha o was detected in the cytoplasm of neuronal cell bodies. Our observations suggest that Go in brain might have a role in membrane signal transduction at synaptic and extrasynaptic sites.
Comparison of time-resolved and -unresolved measurements of deoxyhemoglobin in brain.
Authors: Chance B , Leigh J S , Miyake H , Smith D S , Nioka S , Greenfeld R , Finander M , Kaufmann K , Levy W , Young M
Published: May 2006

Continuous (CW) and pulsed light were used for the noninvasive measurement of hemoglobin oxygenation in tissues. A dual wavelength method of continuous illumination spectroscopy used 760 nm (deoxyhemoglobin peak) and 800 nm (an oxyhemoglobin-deoxyhemoglobin isosbestic point) to measure the kinetics and extent of oxyhemoglobin deoxygenation in brains during mild ischemia/hypoxia. Absorption and scattering were modeled in an artificial milk/yeast blood system, which gave an exponential relationship between absorption and optical path length to a depth of 7 cm. Time-resolved spectroscopy (10-ps resolution) afforded a display of the times and distances of arrival of photons emitted by the cat brain in response to a 10-ps input pulse. The emitted photons rose to a peak in a fraction of a nanosecond and declined exponentially over a few nanoseconds. The half-time of exponential decay corresponds to photon migration over a distance of 4 cm. Exponential light emission continued for several more nanoseconds when the brain was encased by the skull, which plays a key role in prolonging light emission. The exponential decline of light intensity has a value [exp(-microL)], where L is the path length determined from the time/distance scale and mu is the characteristic of the migration of light in the brain. The factor mu is increased by increasing absorption, and mu' = epsilon C where epsilon and C are the Beer-Lambert parameters of extinction coefficient (epsilon) and concentration (C). Thus, deoxyhemoglobin can be quantified in brain tissues.
Monte Carlo simulations of the folding of beta-barrel globular proteins.
Authors: Skolnick J , Kolinski A , Yaris R
Published: May 2006

With the use of dynamic Monte Carlo simulations, the necessary conditions for the collapse from a random-coil denatured state to a structurally unique four-member beta-barrel native state of a model globular protein have been investigated. These systems are free to roam through all of configuration space--both native and nonnative interactions are allowed. The relative importance of hydrophobic and hydrophilic interactions and the presence or absence of statistical bend-forming regions for the formation of a unique native state are examined, and the conditions necessary for a denatured-to-native (and vice versa) conformational transition that is thermodynamically all-or-none and which always results in collapse to the same, four-member beta-barrel are explored. These conditions are found to be a general pattern of hydrophobic/hydrophilic residues that allows the native state to differentiate the interior from the exterior of the protein and the presence of regions that are, at the very least, neutral toward turn formation. The former set of interactions seems to define the mean length of the beta-stretch, and the latter set serves to lock the native state into the lowest free energy state, the native conformation. These folding simulations strongly suggest that the general rules of protein folding are rather robust and that site-specific tertiary interactions are only involved in structural fine tuning. The conditions required for the formation of a structurally unique native state from a manifold of collapsed conformations that are originally quite close in energy is highly suggestive of a mechanism of protein evolution by means of random mutations. The implications of these folding studies for such a mechanism are qualitatively explored.
Feasibility of long-term storage of graded information by the Ca2+/calmodulin-dependent protein kinase molecules of the postsynaptic density.
Authors: Lisman J E , Goldring M A
Published: May 2006

The feasibility of long-term information storage by brain type II Ca2+/calmodulin-dependent protein kinase molecules is explored. Recent evidence indicates that this protein has switch-like properties. Equations are derived showing that a single kinase holoenzyme could form a bistable switch having the stability necessary to encode long-term memory, and that a group of kinase molecules, such as that contained within the postsynaptic density, could form a device capable of storing graded information.
Phosphorylation process induced by epidermal growth factor alters the oncogenic and cellular neu (NGL) gene products.
Authors: Kokai Y , Dobashi K , Weiner D B , Myers J N , Nowell P C , Greene M I
Published: May 2006

The rat neu oncogene encodes a cell surface glycoprotein, p185, that possesses tyrosine kinase activity. The p185 polypeptide exhibits structural similarity to the epidermal growth factor receptor (EGFR) at both the deduced amino acid and nucleic acid level. However, the neu oncogene and the gene encoding the EGFR have been shown to reside on distinct chromosomes. Comparative analysis of the sequences of the normal neu cDNA and of the neu cDNA from neuroblastomas has revealed a single point mutation leading to a valine-to-glutamic acid substitution in the transmembrane anchoring domain. This mutation converts the neu gene to a transforming gene in rodents. In humans, the gene is called ERBB2 (also NGL and HER2), and amplification and over-expression of its products have been detected in certain tumors. The rat embryonal fibroblast cell line (Rat-1) appears to express both EGFR and cellular p185 polypeptides. We have found that EGF stimulates the phosphorylation of p185 in these cells at tyrosine as well as serine and threonine residues in a specific and dose-dependent manner. This activity occurs even though radiolabeled EGF cannot bind to immunopurified p185. The EGF effect is apparently unique since platelet-derived growth factor, insulin, and transforming growth factor beta all fail to phosphorylate p185 at tyrosine. The EGF-induced effect requires interaction of the EGFR and its cognate ligand because cell lines that lack EGFR cannot be shown to phosphorylate p185, even when exposed to large amounts of EGF. Oncogenic rodent p185 and the human p185 homologue ERBB2 that is overexpressed in human breast tumor cells also can be shown to become phosphorylated on tyrosine residues by the action of EGF. Collectively, these data demonstrate that EGF mediates phosphorylation of p185 at tyrosine as well as serine/threonine through cellular kinases by a receptor-specific mechanism.
Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes.
Authors: Bestwick R K , Kozak S L , Kabat D
Published: May 2006

A procedure is described for stably expressing cloned genes at high levels in vertebrate cells and for obtaining these genes in high-titer virus preparations. The process uses retroviral vectors and mixtures of two "packaging cell lines" that incorporate retroviral genomes into virions with different host-range envelopes. In these cocultures, interference barriers to superinfection are overcome, retroviral vectors can replicate in the absence of a transmissible helper virus, and the cells become infected with multiple copies of the provirus that contains the cloned gene. This procedure was used to amplify expression of the membrane glycoprotein that is encoded by Friend spleen focus-forming virus, a retrovirus that is replication defective in other cell cultures. Amplifications were measured at the DNA provirus, RNA, and protein levels. In addition, the human growth hormone gene was inserted into retroviral vectors and we observed amplifications of growth hormone synthesis and secretion. The amplified growth hormone was properly processed as indicated by immunoblot analyses. A vector is described (pSFF) that is exceptionally active in coculture amplification.
Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.
Authors: Fraser C M , Chung F Z , Wang C D , Venter J C
Published: May 2006

By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.
Antibody to sigma 32 cross-reacts with DnaK: association of DnaK protein with Escherichia coli RNA polymerase.
Authors: Skelly S , Fu C F , Dalie B , Redfield B , Coleman T , Brot N , Weissbach H
Published: May 2006

A polyclonal antibody to sigma 32, the heat shock sigma factor, has been used to show the presence of low levels of sigma 32 in Escherichia coli RNA polymerase preparations (E sigma 70), which explains the observed in vitro activity of E sigma 70 towards heat shock genes. The sigma 32 antibody cross-reacts with DnaK, and DnaK has been found associated with purified preparations of both E sigma 70 and the heat shock RNA polymerase, E sigma 32.
Chromosomal location and structure of the operon encoding peptide-chain-release factor 2 of Escherichia coli.
Authors: Kawakami K , Jönsson Y H , Björk G R , Ikeda H , Nakamura Y
Published: May 2006

The prfB gene encodes peptide-chain-release factor 2 of Escherichia coli, which catalyzes translation termination at UGA and UAA codons. The gene, identified by sequencing, is located at the 62-min region of the E. coli chromosome. The prfB gene is followed by an open reading frame encoding a 57,603-Da protein. This downstream open reading frame was identified as herC, a gene defined by a suppressor mutation that restores replication of a ColE1 plasmid mutant. RNA blot hybridization and S1 nuclease protection analyses of in vivo transcripts showed that prfB and herC are cotranscribed into a 2800-base transcript in the counterclockwise direction with respect to the E. coli genetic map. Thus, we refer to the two genes as the prfB-herC operon. Data are presented that suggest that supK, a mutation in Salmonella typhimurium that suppresses UGA termination, is the structural gene for Salmonella release factor 2. Translation control within the prfB-herC operon and the relationship of these genes to a tRNA methyltransferase are discussed.
T-cell epitopes in Pf155/RESA, a major candidate for a Plasmodium falciparum malaria vaccine.
Authors: Kabilan L , Troye-Blomberg M , Perlmann H , Andersson G , Högh B , Petersen E , Björkman A , Perlmann P
Published: May 2006

Immunogens included in a subunit vaccine should contain both B- and T-cell-activating sites to ensure anamnestic responses following reinfection after vaccination as well as antibody-independent cellular immunity. The Plasmodium falciparum antigen Pf155/RESA, a major candidate for a vaccine against the asexual blood stages of this malaria parasite, was investigated for T-cell epitopes in its C-terminal amino acid repeat region, a region known to be conserved in different P. falciparum strains. It was found to contain several related sequences that activated T cells from humans primed to P. falciparum by natural exposure, to proliferation, and/or interferon-gamma release in vitro. T cells from approximately half of the donor group investigated responded to the intact protein, and 65% of these responders also responded to short synthetic peptides, probably representing a small number of partly overlapping T-cell epitopes. Thus, sequences from the C terminus of Pf155 may be suitable constituents of a P. falciparum subunit vaccine and also provide a basis for epitope-specific epidemiological studies relating cellular immune responses in vitro to clinical immunity and P. falciparum endemicity.
Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.
Authors: Ogawa H , Miller D A , Dunn T , Su Y , Burcham J M , Peraino C , Fujioka M , Babcock K , Pitot H C
Published: May 2006

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.
A knotted free minicircle in kinetoplast DNA.
Authors: Ryan K A , Shapiro T A , Rauch C A , Griffith J D , Englund P T
Published: May 2006

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a network containing thousands of minicircles that are topologically interlocked. The minicircle replication intermediates are free molecules that have been released from the network. We report here that one form of free minicircles is a trefoil knot. Identification of this knotted structure is based on its electrophoretic and sedimentation properties, its response to treatments with restriction enzymes or topoisomerase II, and its appearance by electron microscopy. Except for its topology, the knotted minicircle closely resembles a previously described replication intermediate with a unique gap in the newly synthesized L strand.
Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.
Authors: Gruenert D C , Basbaum C B , Welsh M J , Li M , Finkbeiner W E , Nadel J A
Published: May 2006

To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression.
An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes.
Authors: Evans T , Reitman M , Felsenfeld G
Published: May 2006

We have identified a protein present only in erythroid cells that binds to two adjacent sites within an enhancer region of the chicken beta-globin locus. Mutation of the sites, so that binding by the factor can no longer be detected in vitro, leads to a loss of enhancing ability, assayed by transient expression in primary erythrocytes. Binding sites for the erythroid-specific factor (Eryf1) are found within regulatory regions for all chicken globin genes. A strong Eryf1 binding site is also present within the enhancer of at least one human globin gene, and proteins from human erythroid cells (but not HeLa cells) bind to both the chicken and the human sites.
Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae.
Authors: Wickner R B
Published: May 2006

The MAK16 gene was first defined as a gene whose mutation resulted in loss of M1 double-stranded RNA virus-like particles. The mak16-1 mutation also produces temperature-sensitive cell growth. We report here that mak16-1 cells arrest at the nonpermissive temperature in G1 phase, such that they are mating competent. We sequenced the MAK16 gene and found an open reading frame of 306 amino acids encoding a predicted protein of Mr 35,694. Two typical nuclear localization signal sequences were found. MAK16-LacZ fusion proteins that include one of these putative signals entered the nucleus, while unfused beta-galactosidase did not, as judged by subcellular fractionation experiments. In the C-terminal third of the MAK16 open reading frame is an acidic region in which 25 of 41 residues are either glutamate or aspartate. This region contains potential phosphorylation sites for "casein kinases," protein kinases specific for serine or threonine residues in an acidic environment.
Transgenic mice bearing the human c-myc gene activated by an immunoglobulin enhancer: a pre-B-cell lymphoma model.
Authors: Schmidt E V , Pattengale P K , Weir L , Leder P
Published: May 2006

Transgenic mice carrying a fusion gene in which the mouse immunoglobulin enhancer has been inserted into an otherwise normal human c-myc gene develop a narrow spectrum of pre-B-cell lymphomas. Tumor occurrence is correlated with expression of the transgene in organs in which large numbers of pre-B cells predominate. These tumors, which arise stochastically, are virtually all lymphoblastic lymphomas of the pre-B-cell type. Evidently the isolated enhancer targets oncogene expression and tumorigenesis to the early B-cell population in preference to more mature B-cell populations. The transgene also confers enhanced in vitro growth properties on nontransformed pre-B cells as observed in bone marrow cultures derived from transgenic animals. These cultured cells represent a population in which the activating function of c-myc can be uncoupled from secondary oncogenic events occurring in vivo.
Functional reconstitution of the canalicular bile salt transport system of rat liver.
Authors: Ruetz S , Hugentobler G , Meier P J
Published: May 2006

Recent studies have suggested that the canalicular bile salt transport system of rat liver corresponds to a 100-kDa membrane glycoprotein. In the present study we attempted to functionally reconstitute the 100-kDa protein into artificial proteoliposomes. Canalicular membrane proteins were solubilized with octyl glucoside in the presence of asolectin phospholipids. The extracts were treated with preimmune serum or the 100-kDa protein selectively immunoprecipitated with a polyclonal antiserum. Proteins remaining in the supernatant were then incorporated into proteoliposomes by gel-filtration chromatography. Canalicular proteoliposomes containing the 100-kDa protein exhibited transstimulatable taurocholate uptake that could be inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). In contrast, no DIDS-sensitive transstimulatable taurocholate uptake was found in 100-kDa protein-free canalicular proteoliposomes. However, when the immunoprecipitated 100-kDa protein was dissociated from the antibodies and exclusively incorporated into liposomes, reconstitution of DIDS-sensitive transstimulatable and electrogenic taurocholate anion transport was again positive. Although incorporation of solubilized basolateral membrane proteins into liposomes also resulted in a prompt reconstitution of Na+ gradient-driven taurocholate uptake, the anti-100-kDa antibodies had no effects on the reconstituted transport activity of basolateral proteins. Thus, the findings establish that the previously characterized canalicular-specific 100-kDa protein is directly involved in the transcanalicular secretion of bile salts.
Influence of loop residues on the relative stabilities of DNA hairpin structures.
Authors: Senior M M , Jones R A , Breslauer K J
Published: May 2006

We have determined the relative stabilities and melting behaviors of DNA hairpin structures as a function of the nonbonded residues in the loop. The specific family of hairpin structures we investigated in this work is formed by the 16-mer sequence d[CGAACG(X)4CGTTCG], where X is deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine. As shown below, this 16-mer can fold back on itself to form a family of DNA hairpin structures that possess a common hexameric stem duplex and a nonbonded loop of 4 nucleotides. For the hairpin structures investigated in this work, we varied the loop composition from all purine residues to all pyrimidine residues. (Formula: see text). We thermodynamically characterized the relative stabilities and melting profiles of these hairpin structures by a combination of spectroscopic and calorimetric techniques. To establish a thermodynamic "baseline," we also conducted parallel studies on the isolated hexameric duplex d[CGAACG).(CG-TTCG)], which corresponds to the common stem duplex present in each hairpin structure. Our spectroscopic and calorimetric data reveal the following: (i) The hairpin structure with four dT residues in the loop exhibits the highest melting temperature, while the corresponding hairpin structure with four dA residues in the loop exhibits the lowest melting temperature. (ii) The free energy data at 25 degrees C reveal the following order of DNA hairpin stability for the four structures studied here: T loop greater than C loop greater than G loop greater than A loop. In other words, the pyrimidine-looped hairpins of four residues are more stable than the purine-looped hairpins. (iii) The loop-dependent order of hairpin stability is paralleled by a similar trend in the calorimetrically determined transition enthalpies for hairpin disruption. Thus, the enhanced stability of the pyrimidine-looped hairpin structures relative to purine-looped hairpin structures is enthalpic in origin. To develop insight into the molecular basis for the thermodynamic differences, proton NMR spectroscopy was used to probe for structural disparities between the most stable hairpin structure (T loop) and the least stable hairpin structure (A loop). Two-dimensional nuclear Overhauser enhancement spectroscopy revealed connectivities between the residues in the stem duplexes of both hairpin structures that are consistent with B-form DNA. In addition, the nonbonded residues in both the T and A loops exhibited the same connectivity patterns. However, on the 5' side of the stem-loop junction, the T-loop residue exhibited a connectivity with the adjacent base pair of the stem duplex that is not observed for the corresponding A-loop residue. This difference in connectivities at the stem-loop junction may provide a structural basis for our observation that the T-looped hairpin structure is more stable than the corresponding A-looped hairpin structure.
Selective modulation of protein kinase isozymes by the site-selective analog 8-chloroadenosine 3',5'-cyclic monophosphate provides a biological means for control of human colon cancer cell growth.
Authors: Ally S , Tortora G , Clair T , Grieco D , Merlo G , Katsaros D , Ogreid D , Døskeland S O , Jahnsen T , Cho-Chung Y S
Published: May 2006

Differential expression of type I and type II cAMP-dependent protein kinase isozymes has been linked to growth regulation and differentiation. We examined the expression of protein kinase isozymes in the LS 174T human colon cancer cell line during 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP)-induced growth inhibition. Two species of RII (the regulatory subunit of protein kinase type II) with apparent Mr 52,000 (RII52) and Mr 56,000 (RII56) and a single species of RI (the regulatory subunit of protein kinase type I) with Mr 48,000 were identified in the cancer cells. RI and both forms of RII were covalently labeled with 8-azidoadenosine 3',5'-cyclic [32P]monophosphate, and two anti-RII antibodies that exclusively recognize either RII52 or RII56 resolved two forms of the RII receptors. 8-Cl-cAMP treatment induced a decrease of RI and an increase of both RII52 and RII56 in the cytosols of cancer cells and rapid translocation (within 10 min) of RII52 from the cytosol to nucleus. 8-Cl-cAMP caused transcriptional activation of the RII52 receptor gene and inactivation of the RI receptor gene. It also exhibited high-affinity site-1-selective binding to the purified preparations of both RII receptor proteins. Thus, differential regulation of various forms of cAMP receptor proteins is involved in 8-Cl-cAMP-induced regulation of cancer cell growth, and nuclear translocation of RII52 receptor protein appears to be an early event in such differential regulation.
Self-organization of the fluid mosaic of charged channel proteins in membranes.
Authors: Fromherz P
Published: May 2006

Electrically charged ion channels in a fluid membrane may form dissipative structures driven by a concentration gradient of salt. On a molecular level the effect is due to dissipative attractive forces; the channel currents induce local gradients of the membrane potential that interact with the protein charge. Self-organization by "charged channel condensation" is treated on a phenomenological level: Smoluchowski's equation describing diffusion and drift of the membrane proteins and Kelvin's equation describing the dynamics of the membrane potential are considered as a coupled system of equations. The patterns of the two morphogens, the membrane protein and the membrane potential, are controlled by global parameters--the average density of charged channels, the level of their reversal potential, and the size of the membrane.
Independent photocycles of the spectrally distinct forms of bacteriorhodopsin
Authors: Dancsházy Zs. , Govindjee R. , Ebrey T. G.
Published: May 2006

Time-resolved, flash-induced difference absorbance spectra (300-700 nm) at pH 10.5 and 5°C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (M
f
and M
s
, respectively) and R intermediate are reported. The main distinguishing features are as follows: For M
f
, Δ
A
max
= 412 nm, a shoulder at 436 nm, no absorbance change at 350 nm; Δ
A
min
= 565 nm; Δ
A
412
/Δ
A
565
= 0.85. For M
s
, Δ
A
max
= 412 nm, a shoulder at 386 nm; Δ
A
min
= 575 nm; Δ
A
412
/Δ
A
575
= 0.6. For R, Δ
A
max
= 336 and 350 nm (double peak), smaller peaks at 386 and 412 nm; Δ
A
min
= 585 nm; Δ
A
350
/Δ
A
585
= 0.2. The different difference spectra for M
f
and M
s
provide direct evidence that these species, initially identified by their kinetics, are physically distinct. With fast transient absorption spectroscopy, it was shown that R may form very fast, perhaps faster than the L intermediate decays. On the basis of the different bleaching peaks for M
f
and M
s
, we propose that M
f
and M
s
are in independent photocycles formed from slightly different forms of bacteriorhodopsin. R may also be in a different photocycle. The different forms of bacteriorhodopsin are probably in dynamic equilibrium with their ratios, controlled by pH and temperature.