bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer.
Authors: Wang J , Smerdon S J , Jäger J , Kohlstaedt L A , Rice P A , Friedman J M , Steitz T A
Published: May 2006

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.
A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.
Authors: Ragan M A , Bird C J , Rice E L , Gutell R R , Murphy C A , Singh R K
Published: May 2006

A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups.
A gene phylogeny of the red algae (Rhodophyta) based on plastid rbcL.
Authors: Freshwater D W , Fredericq S , Butler B S , Hommersand M H , Chase M W
Published: May 2006

A phylogeny for the Rhodophyta has been inferred by parsimony analysis of plastid rbcL sequences representing 81 species, 68 genera, 38 families, and 17 orders of red algae; rbcL encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Levels of sequence divergence among species, genera, and families are high in red algae, typically much greater than those reported for flowering plants. The Rhodophyta traditionally consists of one class, Rhodophyceae, and two subclasses, Bangiophycidae and Florideophycidae. The Bangiophycidae with three orders (Porphyridiales, Compsopogonales, and Bangiales) appears to be polyphyletic, and the Florideophycidae with 17 orders is monophyletic in this study. The current classification of the Florideophycidae based on ultrastructure of pit connections is supported. With the exception of the Rhodogorgonales, which appears to be misplaced, orders with one or two pit-plug cap layers (Hildenbrandiales, Corallinales, Acrochaetiales, Palmanales, Batrachospermales, and Nemaliales) terminate long branches of basal position within Florideophycidae in the most parsimonious rbcL tree. Orders that lack typical cap layers but possess a cap membrane are resolved as a monophyletic clade sister to the Ahnfeltiales. The large order Gigartinales, which is distributed among five rbcL clades, is polyphyletic. Families that possess typical carrageenan in their cell walls are resolved as a terminal clade containing two family complexes centered around the Solieriaceae and Gigartinaceae.
Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII.
Authors: Yee V C , Pedersen L C , Le Trong I , Bishop P D , Stenkamp R E , Teller D C
Published: May 2006

Mechanical stability in many biological materials is provided by the crosslinking of large structural proteins with gamma-glutamyl-epsilon-lysyl amide bonds. The three-dimensional structure of human recombinant factor XIII (EC 2.3.2.13 zymogen; protein-glutamine:amine gamma-glutamyltransferase a chain), a transglutaminase zymogen, has been solved at 2.8-A resolution by x-ray crystallography. This structure shows that each chain of the homodimeric protein is folded into four sequential domains. A catalytic triad reminiscent of that observed in cysteine proteases has been identified in the core domain. The amino-terminal activation peptide of each subunit crosses the dimer interface and partially occludes the opening of the catalytic cavity in the second subunit, preventing substrate binding to the zymogen. A proposal for the mechanism of activation by thrombin and calcium is made that details the structural events leading to active factor XIIIa'.
The bifunctional iron-responsive element binding protein/cytosolic aconitase: the role of active-site residues in ligand binding and regulation.
Authors: Philpott C C , Klausner R D , Rouault T A
Published: May 2006

The iron-responsive element binding protein/cytosolic aconitase functions as either an RNA binding protein that regulates the uptake, sequestration, and utilization of iron or an enzyme that interconverts citrate and isocitrate. These mutually exclusive functions are regulated by changes in cellular iron levels. By site-directed mutagenesis we show that (i) ligation of a [4Fe-4S] cluster is necessary to inactivate RNA binding and activate enzyme function in vivo, (ii) three of four arginine residues of the aconitase active site participate in RNA binding, and (iii) aconitase activity is not required for iron-mediated regulation of RNA binding.
Attention: the mechanisms of consciousness.
Authors: Posner M I
Published: May 2006

A number of recent papers and books discuss theoretical efforts toward a scientific understanding of consciousness. Progress in imaging networks of brain areas active when people perform simple tasks may provide a useful empirical background for distinguishing conscious and unconscious information processing. Attentional networks include those involved in orienting to sensory stimuli, activating ideas from memory, and maintaining the alert state. This paper reviews recent findings in relation to classical issues in the study of attention and anatomical and physical theories of the nature of consciousness.
Nitric oxide as a mediator of oxidant lung injury due to paraquat.
Authors: Berisha H I , Pakbaz H , Absood A , Said S I
Published: May 2006

At low concentrations, nitric oxide is a physiological transmitter, but in excessive concentrations it may cause cell and tissue injury. We report that in acute oxidant injury induced by the herbicide paraquat in isolated guinea pig lungs, nitric oxide synthesis was markedly stimulated, as evidenced by increased levels of cyclic GMP in lung perfusate and of nitrite and L-citrulline production in lung tissue. All signs of injury, including increased airway and perfusion pressures, pulmonary edema, and protein leakage into the airspaces, were dose-dependently attenuated or totally prevented by either NG-nitro-L-arginine methyl ester or N omega-nitro-L-arginine, selective and competitive inhibitors of nitric oxide synthase. Protection was reversed by excess L-arginine but not by its enantiomer D-arginine. When blood was added to the lung perfusate, the paraquat injury was moderated or delayed as it was when paraquat was given to anesthetized guinea pigs. The rapid onset of injury and its failure to occur in the absence of Ca2+ suggest that constitutive rather than inducible nitric oxide synthase was responsible for the stimulated nitric oxide synthesis. The findings indicate that nitric oxide plays a critical role in the production of lung tissue injury due to paraquat, and it may be a pathogenetic factor in other forms of oxidant tissue injury.
p53 induction is associated with neuronal damage in the central nervous system.
Authors: Sakhi S , Bruce A , Sun N , Tocco G , Baudry M , Schreiber S S
Published: May 2006

The p53 tumor-suppressor gene encodes a growth-regulatory protein that has been implicated in programmed cell death. To investigate the possible role of p53 in neuronal death, we studied p53 expression associated with excitotoxicity in the adult rat brain. Within hours of systemic administration of the glutamate analogue kainic acid, p53 mRNA levels were increased in neurons exhibiting morphological features of damage within kainate-vulnerable brain regions. A similar distribution was found for neurons exhibiting DNA damage as evidenced by in situ end-labeling of fragmented DNA. Pretreatment with the protein synthesis inhibitor cycloheximide prevented both kainate-mediated p53 induction and neuronal damage. The distinctive pattern of excitotoxin-mediated p53 expression suggests that p53 induction is a marker of irreversible injury in postmitotic cells of the central nervous system and could have functional significance in determining selective neuronal vulnerability.
A human oncogene of the RAS superfamily unmasked by expression cDNA cloning.
Authors: Chan A M , Miki T , Meyers K A , Aaronson S A
Published: May 2006

As an approach to identify human oncogenes, we generated an expression cDNA library from an ovarian carcinoma line. A potent transforming gene was detected by transfection analysis and identified as TC21, a recently cloned member of the RAS gene superfamily. A single point mutation substituting glutamine for leucine at position 72 was shown to be responsible for activation of transforming properties. While the cDNA clone possessed high transforming activity, the ovarian tumor genomic DNA, which contained the mutated TC21 allele, failed to induce transformed foci. Thus, expression cDNA cloning made it possible to identify and isolate a human oncogene that has evaded detection by conventional approaches.
Clustering of Ca2+ channels and Ca(2+)-activated K+ channels at fluorescently labeled presynaptic active zones of hair cells.
Authors: Issa N P , Hudspeth A J
Published: May 2006

Electrical resonance, which in some hair cells provides a mechanism for frequency tuning, is mediated by clusters of Ca2+ channels and Ca(2+)-activated K+ channels that have been proposed to occur at presynaptic active zones. To localize Ca2+ channels on the cellular surface, we loaded hair cells from the frog's sacculus with the Ca2+ indicator fluo-3 and imaged them by fluorescence confocal microscopy. When a cell was depolarized, we observed on its basolateral surface several foci of transiently enhanced fluorescence due to local Ca2+ influx. After protracted recording, each cell displayed on average 18 brightly and permanently fluorescent spots at the same positions. We mapped these spots in four hair cells and compared their locations with those of presynaptic active zones, as determined from transmission electron micrographs of serial sections through the same cells. The results demonstrated that enhanced fluo-3 fluorescence marks active zones. Measurement of currents through membrane patches at fluorescently labeled active zones demonstrated that both voltage-activated Ca2+ channels and Ca(2+)-activated K+ channels occur there. These results confirm that the ion channels involved in electrical tuning and synaptic transmission by hair cells cluster together at presynaptic active zones.
Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium.
Authors: Hansen G , Das A , Chilton M D
Published: May 2006

Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system. In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction. A mutation of virG, virGN54D (which codes for a Asn-54-->Asp amino acid change in the product), causes constitutive expression of other vir genes independent of virA. We have investigated whether providing Agrobacterium with a plasmid containing virGN54D augments the efficiency of transfer of the T-DNA (transferred DNA). For both tobacco and cotton, we observed an enhancement of transformation efficiency when the inciting Agrobacterium strain carries the virGN54D mutation. We also tested whether supplying Agrobacterium with a similar plasmid containing wild-type virG affects the efficiency of T-DNA transfer. An intermediate efficiency was observed when this plasmid was employed. Using a beta-glucuronidase (GUS) reporter gene to assess transient expression of T-DNA after transfer to tobacco and maize tissues, we observed a higher frequency of GUS-expressing foci after inoculation with Agrobacterium strains carrying virGN54D than with Agrobacterium carrying the wild-type virG. Gene-transfer efficiency to maize by an octopine strain was greatly improved upon introduction of virGN54D. Multiple copies of wild-type virG were equally effective in promoting transient expression efficiency in tobacco but were virtually ineffective in maize. We propose the use of virGN54D to improve the efficiency of Agrobacterium-mediated transformation, especially for recalcitrant plant species.
Glycated tau protein in Alzheimer disease: a mechanism for induction of oxidant stress.
Authors: Yan S D , Chen X , Schmidt A M , Brett J , Godman G , Zou Y S , Scott C W , Caputo C , Frappier T , Smith M A
Published: May 2006

The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGE-recombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.
Naturally occurring active N-domain of human angiotensin I-converting enzyme.
Authors: Deddish P A , Wang J , Michel B , Morris P W , Davidson N O , Skidgel R A , Erdös E G
Published: May 2006

Angiotensin I-converting enzyme (ACE, kininase II) is a single-chain protein containing two active site domains (named N- and C-domains according to position in the chain). ACE is bound to plasma membranes by its C-terminal hydrophobic transmembrane anchor. Ileal fluid, rich in ACE activity, obtained from patients after surgical colectomy was used as the source. Column chromatography, including modified affinity chromatography on lisinopril-Sepharose, yielded homogeneous ACE after only a 45-fold purification. N-terminal sequencing of ileal ACE and partial sequencing of CNBr fragments revealed the presence of an intact N terminus but only a single N-domain active site, ending between residues 443 and 559. Thus, ileal-fluid ACE is a unique enzyme differing from the widely distributed two-domain somatic enzyme or the single C-domain testicular (germinal) ACE. The molecular mass of ileal ACE is 108 kDa and when deglycosylated, the molecular mass is 68 kDa, indicating extensive glycosylation (37% by weight). In agreement with the results reported with recombinant variants of ACE, the ileal enzyme is less Cl(-)-dependent than somatic ACE; release of the C-terminal dipeptide from a peptide substrate was optimal in only 10 mM Cl-. In addition to hydrolyzing at the C-terminal end of peptides, ileal ACE efficiently cleaved the protected N-terminal tripeptide from the luteinizing hormone-releasing hormone and its congener 6-31 times faster, depending on the Cl- concentration, than the C-domain in recombinant testicular ACE. Thus we have isolated an active human ACE consisting of a single N-domain. We suggest that there is a bridge section of about 100 amino acids between the active N- and C-domains of somatic ACE where it may be proteolytically cleaved to liberate the active N-domain. These findings have potential relevance and importance in the therapeutic application of ACE inhibitors.
Follicular origin of epidermal papillomas in v-Ha-ras transgenic TG.AC mouse skin.
Authors: Hansen L A , Tennant R W
Published: May 2006

A follicular origin for some skin tumors has been hypothesized in both humans and animal models. Because of its rapid and sensitive response to tumor promoter treatment, a v-Ha-ras transgenic (TG.AC) mouse line was used to determine the origins of epidermal papillomas. Using histological studies and transgene expression as a marker for papilloma development, we determined that pedunculated papillomas arose from focal hyperplasias of the permanent portion of the follicular epithelium in phorbol 12-myristate 13-acetate-treated TG.AC mouse skin. Damage to the hair follicle by depilation was also sufficient to induce papillomas that were histologically indistinguishable from those produced by chemical exposure. Identification of the cellular origins of papillomas in this transgenic mouse model will allow for an analysis of the role of the hair follicle and hair cycle-associated signaling in tumor development.
Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein.
Authors: Louis J M , Nashed N T , Parris K D , Kimmel A R , Jerina D M
Published: May 2006

Upon renaturation, the polyprotein MBP-delta TF-Protease-delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (delta TF) and the polymerase (delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps. The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-delta Pol, which has enzymatic activity comparable to that of the mature enzyme. Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus. The rate of increase in enzymatic activity is identical to that of the appearance of MBP-delta TF and the disappearance of the MBP-delta TF-Protease-delta Pol. Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration. The reaction is competitively inhibited by pepstatin A and has a pH rate profile similar to that of the mature enzyme. These results and molecular modeling studies are discussed in terms of a mechanism in which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.
A synthetic peptide from the third hypervariable region of major histocompatibility complex class II beta chain as a vaccine for treatment of experimental autoimmune encephalomyelitis.
Authors: Topham D J , Nag B , Arimilli S , Sriram S
Published: May 2006

Experimental autoimmune encephalomyelitis (EAE) is a class II major histocompatibility complex (MHC)-restricted, T-cell-mediated, demyelinating autoimmune disease of the central nervous system and represents a model for human multiple sclerosis. The present study demonstrates that vaccination of SJL/J mice with an 18-amino acid synthetic peptide from the third hypervariable region of the murine class II MHC IAs beta chain (IAs beta 58-75; 18-mer peptide) is capable of eliciting auto-anti-IAs antibodies specific for the IAs beta chain and preventing and treating EAE. A similar approach may be useful in the treatment of human autoimmune diseases in which susceptibility is linked to class II MHC genes.
Visualizing hippocampal synaptic function by optical detection of Ca2+ entry through the N-methyl-D-aspartate channel.
Authors: Malinow R , Otmakhov N , Blum K I , Lisman J
Published: May 2006

Fura-2 and imaging technology were used to detect intracellular Ca2+ changes in CA1 pyramidal cells in hippocampal slices. During focal synaptic stimulation, one or more highly localized regions of Ca2+ elevation (hot spots) were detected in the dendrites. Ca2+ spread from the center of hot spots with properties consistent with diffusion. Several lines of evidence indicate that these hot spots were due to Ca2+ entry through N-methyl-D-aspartate synaptic channels. The spatial and temporal resolution of the method was sufficient to detect the response of single hot spots to single stimuli, thus providing a real-time method for monitoring local synaptic activity. Using this method, we show that synapses on the same dendrite differ in their probability of response and in their facilitation properties.
A regulatory system for use in gene transfer.
Authors: Wang Y , O'Malley B W , Tsai S Y , O'Malley B W
Published: May 2006

We recently have demonstrated that a C-terminal deletion mutant of the human progesterone receptor (hPRB891) fails to bind to progesterone but can bind RU 486 (Mifepristone) and other progesterone antagonists. Most significantly, this mutant receptor activates transcription of a reporter gene containing the progesterone response element in the presence of these antagonists. Taking advantage of this finding and the modular nature of functional domains of steroid receptors, we constructed a chimeric regulator (pGL-VP) by fusing the ligand-binding domain of human progesterone receptor hPRB891 to the yeast transcriptional activator GAL4 DNA-binding domain and the herpes simplex virus protein VP16 activation domain. We demonstrated that this chimeric regulator activates target genes containing the GAL4-binding sites in transient transfection assays in response to RU 486. In addition, this regulatory system has been validated by ex vivo transplantation of a stable cell line containing both the regulator and a reporter gene into rats. The dosage of RU 486 used is significantly lower than that required for antagonizing progesterone action. The gene-switch system reported here represents a regulatory system, which could be applicable for gene-transfer studies involving animals, as well as humans, in which the delivered gene(s) can be specifically turned on/off in response to an exogenous compound.
Electron microscopic studies of the interaction between a Bacillus subtilis alpha/beta-type small, acid-soluble spore protein with DNA: protein binding is cooperative, stiffens the DNA, and induces negative supercoiling.
Authors: Griffith J , Makhov A , Santiago-Lara L , Setlow P
Published: May 2006

DNA within spores of Bacillus subtilis is complexed with a group of alpha/beta-type small acid-soluble spore proteins (alpha/beta-type SASPs), which have almost identical primary sequences and DNA binding properties. Here electron microscopic and cyclization studies were carried out on alpha/beta-type SASP-DNA complexes. When an alpha/beta-type SASP was incubated with linear DNA, the protein bound cooperatively, forming a helical coating 6.6 +/- 0.4 nm wide with a 2.9 +/- 0.3 nm periodicity. alpha/beta-Type SASP binding to an 890-bp DNA was weakest at an (A+T)-rich region that was highly bent, but binding eliminated the bending. alpha/beta-Type SASP binding did not alter the rise per bp in DNA but greatly increased the DNA stiffness as measured by both electron microscopic and cyclization assays. Addition of alpha/beta-type SASPs to negatively supertwisted DNA led to protein binding without significant alteration of the plectonemically interwound appearance of the DNA. Addition of alpha/beta-type SASPs to relaxed or nicked circular DNA led to molecules that by electron microscopy appeared similar to supertwisted DNA. The introduction of negative supertwists in nicked circular DNA by alpha/beta-type SASPs was confirmed by ligation of these molecules followed by topoisomer analyses using agarose gel electrophoresis.
NMR structure of a biologically active peptide containing the RNA-binding domain of human immunodeficiency virus type 1 Tat.
Authors: Mujeeb A , Bishop K , Peterlin B M , Turck C , Parslow T G , James T L
Published: May 2006

The Tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific RNA element on nascent viral transcripts. Binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. Here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 Tat basic domain linked to the core regulatory domain of another lentiviral Tat--i.e., that from equine infectious anemia virus. Circular dichroism and two-dimensional proton NMR studies of this hybrid peptide indicate that the Tat basic domain forms a stable alpha-helix, whereas the adjacent regulatory sequence is mostly in extended form. These findings suggest that the tendency to form stable alpha-helices may be a common property of arginine- and lysine-rich RNA-binding domains.
Lambda foo: a lambda phage vector for the expression of foreign proteins.
Authors: Maruyama I N , Maruyama H I , Brenner S
Published: May 2006

This work describes a lambda phage expression system, lambda foo, that produces foreign proteins fused to the surface of the virus particle. The lambda foo vector has multiple cloning sites for the insertion of a foreign DNA fragment and color selection for recombinants. Foreign proteins are fused to the C terminus of a truncated phage tail protein, pV, by a peptide linker. Conditional chain termination allows the assembly and fusion of multisubunit proteins. We have attached the complete Escherichia coli beta-galactosidase and the plant Bauhinia purpurea agglutinin by cloning their genes into the vector. The constructs express functionally active proteins on the phage particle surface and have been purified by affinity chromatography with an antibody for beta-galactosidase and a mucin as a ligand for Bauhinia purpurea agglutinin.
Mos oncogene product associates with kinetochores in mammalian somatic cells and disrupts mitotic progression.
Authors: Wang X M , Yew N , Peloquin J G , Vande Woude G F , Borisy G G
Published: May 2006

The mos protooncogene has opposing effects on cell cycle progression. It is required for reinitiation of meiotic maturation and for meiotic progression through metaphase II, yet it is an active component of cytostatic factor. mos is a potent oncogene in fibroblasts, but high levels of expression are lethal. The lethality of mos gene expression in mammalian cells could be a consequence of a blockage induced by its cytostatic factor-related activity, which may appear at high dosage in mitotic cells. We have directly tested whether expression of the Mos protein can block mitosis in mammalian cells by microinjecting a fusion protein between Escherichia coli maltose-binding protein and Xenopus c-Mos into PtK1 epithelial cells and analyzing the cells by video time-lapse and immunofluorescence microscopy. Time-course analyses showed that Mos blocked mitosis by preventing progression to a normal metaphase. Chromosomes frequently failed to attain a bipolar orientation and were found near one pole. Injection of a kinase-deficient mutant Mos had no effect on mitosis, indicating that the blockage of mitotic progression required Mos kinase activity. Antitubulin immunostaining of cells blocked by Mos showed that microtubules were present but that spindle morphology was abnormal. Immunostaining for the Mos fusion protein showed that both wild-type and kinase mutant proteins localized at the kinetochores. Our results suggest that mitotic blockage by Mos may result from an action of the Mos kinase on the kinetochores, thus increasing chromosome instability and preventing normal congression.
Monophyletic origin of beta-division proteobacterial endosymbionts and their coevolution with insect trypanosomatid protozoa Blastocrithidia culicis and Crithidia spp.
Authors: Du Y , Maslov D A , Chang K P
Published: May 2006

Some trypanosomatid protozoa (order Kinetoplastida) are well known to harbor bacterial endosymbionts. Their phylogenetic positions and evolutionary relationships with the hosts were deduced by comparing the rRNA gene sequences. Earlier, we observed that these symbionts from three Crithidia spp. are identical and are closely related to Bordetella bronchiseptica. We have now sequenced the genes of another endosymbiont and the host protozoan Blastocrithidia culicis. The 16S rRNA genes of the Blastocrithidia and Crithidia symbionts share approximately 97% identity and form a distinct group, branching off the B. bronchiseptica lineage in the beta-division of Proteobacteria. Comparison of their secondary structures in the stem regions suggests compensatory mutations of the symbiont sequences, contributing to their biased base transitions from G to A and C to T. Two putative genes encoding tRNA(Ile) and tRNA(Ala) are highly conserved in the otherwise variable internal transcribed spacer region. Comparisons of the host rRNA gene sequences suggest that the symbiont-containing Crithidia and Blastocrithidia are more akin to each other than to other trypanosomatids. The evidence suggests that Blastocrithidia and Crithidia symbionts descend from a common ancestor, which had presumably entered an ancestral host and thence coevolved with it into different species. We therefore propose naming the symbionts Kinetoplastibacterium blastocrithidii and Kinetoplastibacterium crithidii.
Parvovirus particles as platforms for protein presentation.
Authors: Miyamura K , Kajigaya S , Momoeda M , Smith-Gill S J , Young N S
Published: May 2006

Empty capsids of the human pathogenic parvovirus B19 can be produced in a baculovirus system. B19 capsids are composed mainly of major capsid protein (VP2) and a small amount of minor capsid protein (VP1); VP1 is identical to VP2 but contains an additional 227-aa N-terminal region ("unique" region). A portion of that region of VP1 is external to the capsid, and VP1 is not required for capsid formation. We substituted the unique region with a sequence encoding the 147 aa of hen egg white lysozyme (HEL) and constructed recombinant baculoviruses with variable amounts of retained VP1 sequence joined to the VP2 backbone. After cotransfection with VP2 baculovirus and expression in insect cells, capsids were purified by density sedimentation. Purified recombinant capsids contained HEL. External presentation of HEL was demonstrated by immunoprecipitation, ELISA, and immune electron microscopy using anti-lysozyme monoclonal antibodies or specific rabbit antisera. Empty particles showed enzymatic activity in a micrococcal cell wall digestion assay. Rabbits inoculated with capsids made antibodies to HEL. Intact heterologous protein can be incorporated in B19 particles and presented on the capsid surface, properties that may be useful in vaccine development, cell targeting, and gene therapy.
Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.
Authors: Bivin D B , Ue K , Khoroshev M , Morales M
Published: May 2006

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site--the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product--in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl- for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of Cys-707, or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.
Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein.
Authors: Quinn T P , Tweedy N B , Williams R W , Richardson J S , Richardson D C
Published: May 2006

How an amino acid sequence encodes the information necessary for a protein to adopt a unique tertiary structure remains unresolved. We are addressing this problem by designing "from scratch" protein molecules that will adopt predetermined three-dimensional structures. Based on this strategy, two identical four-stranded beta-sheets were designed to dimerize and form a beta-sandwich protein, called betadoublet. A synthetic gene encoding half the beta-sandwich protein was expressed in Escherichia coli, and the protein was purified to homogeneity. Biophysical characterization of betadoublet in aqueous solution demonstrated that the disulfide formed between the two sheets and that the dimer was a compact unaggregated globular protein, consisting predominantly of beta-sheet and stable to thermal denaturation. It has some backbone amide protons whose exchange is slow enough to be measured by NMR but binds more of the dye 1-anilinonaphthalene-8-sulfonate than a well-folded protein.
P-selectin induces the expression of tissue factor on monocytes.
Authors: Celi A , Pellegrini G , Lorenzet R , De Blasi A , Ready N , Furie B C , Furie B
Published: May 2006

P-selectin on activated platelets and stimulated endothelial cells mediates cell adhesion with monocytes and neutrophils. Since activated platelets induce tissue factor on mononuclear leukocytes, we examined the effect of P-selectin on the expression of tissue factor activity in monocytes. Purified P-selectin stimulated tissue factor expression on mononuclear leukocytes in a dose-dependent manner. Chinese hamster ovary (CHO) cells expressing P-selectin stimulated tissue factor procoagulant activity in purified monocytes, whereas untransfected CHO cells and CHO cells expressing E-selectin did not. Anti-P-selectin antibodies inhibited the effects of purified P-selectin and CHO cells expressing P-selectin on monocytes. Incubation of CHO cells expressing P-selectin with monocytes leads to the development of tissue factor mRNA in monocytes and to the expression of tissue factor antigen on the monocyte surface. These results indicate that P-selectin upregulates the expression of tissue factor on monocytes as well as mediates the binding of platelets and endothelial cells with monocytes and neutrophils. The binding of P-selectin to monocytes in the area of vascular injury may be a component of a mechanism that initiates thrombosis.
Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase.
Authors: Estruch J J , Kadwell S , Merlin E , Crossland L
Published: May 2006

A calcium-dependent calmodulin-independent protein kinase (CDPK) has been cloned from maize (Zea mays). The sequence predicts a 550-amino acid (predicted molecular mass is 60 kDa) protein with two major functional domains: an N-terminal catalytic domain highly homologous to protein kinases and a C-terminal domain resembling calmodulins. Northern analysis shows that the expression of the maize CDPK gene is pollen specific and that its transcription is restricted to late stages of pollen development. Western blots reveal a major abundance of CDPK protein at the stage of pollen germination. In vitro germination and pollen tube growth are impaired upon addition of a calmodulin antagonist (calmidazolium), CDPK inhibitors (W-7), and antisense oligonucleotides directed against CDPK mRNA. These observations indicate that the function of the pollen-specific maize CDPK protein is required for germination and pollen tube growth.
Adeno-associated virus vectors preferentially transduce cells in S phase.
Authors: Russell D W , Miller A D , Alexander I E
Published: May 2006

Vectors based on adeno-associated virus can stably transfer genes by chromosomal integration in recipient cells. In this study we have infected stationary and dividing primary human fibroblast cultures with adeno-associated virus vectors encoding alkaline phosphatase and neomycin phosphotransferase. We find that the transduction frequency of S phase cells is about 200 times that of non-S phase cells. However, neither S phase nor mitosis is essential for transduction. Single-stranded vector genomes survive in stationary cultures and can be recruited for transduction by stimulating these cultures to divide. Stable transductants contain randomly integrated vector sequences. These findings have important implications for the use of adeno-associated virus vectors in gene therapy.
Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels.
Authors: Rundström N , Schmieden V , Betz H , Bormann J , Langosch D
Published: May 2006

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 >> 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.
Femtosecond photodichroism studies of isolated photosystem II reaction centers.
Authors: Wiederrecht G P , Seibert M , Govindjee  , Wasielewski M R
Published: May 2006

Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 beta-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Qy transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.
Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein.
Authors: Imai S , Koizumi S , Sugiura M , Tokunaga M , Uemura Y , Yamamoto N , Tanaka S , Sato E , Osato T
Published: May 2006

In 1000 primary gastric carcinomas, 70 (7.0%) contained Epstein-Barr virus (EBV) genomic sequences detected by PCR and Southern blots. The positive tumors comprised 8 of 9 (89%) undifferentiated lymphoepithelioma-like carcinomas, 27 of 476 (5.7%) poorly differentiated adenocarcinomas, and 35 of 515 (6.8%) moderately to well-differentiated adenocarcinomas. In situ EBV-encoded small RNA 1 hybridization and hematoxylin/eosin staining in adjacent sections showed that the EBV was present in every carcinoma cell but was not significantly present in lymphoid stroma and in normal mucosa. Two-color immunofluorescence and hematoxylin/eosin staining in parallel sections revealed that every keratin-positive epithelial malignant cell expressed EBV-determined nuclear antigen 1 (EBNA1) but did not significantly express CD45+ infiltrating leukocytes. A single fused terminal fragment was detected in each of the EBNA1-expressing tumors, thereby suggesting that the EBV-carrying gastric carcinomas represent clonal proliferation of cells infected with EBV. The carcinoma cells had exclusively EBNA1 but not EBNA2, -3A, -3B, and -3C; leader protein; and latent membrane protein 1 because of methylation. The patients with EBV-carrying gastric carcinoma had elevated serum EBV-specific antibodies. The EBV-specific cellular immunity was not significantly reduced; however, the cytotoxic T-cell target antigens were not expressed. These findings strongly suggest a causal relation between a significant proportion of gastric carcinoma and EBV, and the virus-carrying carcinoma cells may evade immune surveillance.
The alpha/beta sheath and its cytoplasmic tyrosines are required for signaling by the B-cell antigen receptor but not for capping or for serine/threonine-kinase recruitment.
Authors: Williams G T , Peaker C J , Patel K J , Neuberger M S
Published: May 2006

The B-cell antigen receptor is composed of membrane immunoglobulin sheathed by an alpha/beta heterodimer. The complex is noncovalently associated with protein kinase activity, and crosslinking of the receptor leads to capping and transmembrane signaling. Here we show that the sheath is not necessary either for this capping or for the association of membrane immunoglobulin with the detergent-insoluble cytoskeletal fraction that occurs following crosslinking. It is also not required for association of membrane immunoglobulin with a casein-kinase-like serine/threonine kinase. The sheath is essential, however, for transmembrane signaling. Provision of just the cytoplasmic domain of the beta sheath polypeptide to a mutant, unsheathed IgM molecule was sufficient to restore full signaling capability as judged by the phosphorylation of a variety of cellular proteins, including the B-cell-specific transmembrane protein CD22. This signaling was destroyed by mutating one of the tyrosines in the beta cytoplasmic domain. These results not only suggest that receptor signaling is mediated through phosphorylation of the tyrosines in the sheath's cytoplasmic domains but, together with previous work, indicate that different motifs within the sheath mediate presentation and signaling.
Internal Ca2+ mobilization is altered in fibroblasts from patients with Alzheimer disease.
Authors: Ito E , Oka K , Etcheberrigaray R , Nelson T J , McPhie D L , Tofel-Grehl B , Gibson G E , Alkon D L
Published: May 2006

The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca(2+)-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphate-mediated release of intracellular Ca2+.
Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates.
Authors: Ioffe E , Stanley P
Published: May 2006

Eukaryotic cells require N-linked carbohydrates for survival. However, the biosynthetic intermediate Man5GlcNAc2Asn, in place of mature N-linked structures, allows glycoprotein synthesis and somatic cell growth to proceed normally. To determine whether the same would be true in a complex biological situation, the gene Mgat-1 was disrupted by homologous recombination in embryonic stem cells and transmitted to the germ line. The Mgat-1 gene encodes N-acetylglucosaminyltransferase I [GlcNAc-TI; alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; UDP-N-acetyl-D-glucosamine:glycoprotein (N-acetyl-D-glucosamine to alpha-D-mannosyl-1,3-(R1)-beta-D-mannosyl-R2) beta-1,2-N-acetyl-D-glucosaminyltransferase, EC 2.4.1.101], the transferase that initiates synthesis of hybrid and complex N-linked carbohydrates from Man5GlcNAc2Asn. Mice lacking GlcNAc-TI activity did not survive to term. Biochemical and morphological analyses of embryos from 8.5 to 13.5 days of gestation showed that Mgat-1-/-embryos are developmentally retarded, most noticeably in neural tissue, and die between 9.5 and 10.5 days of development.