bluuebunny/crossref_metadata_2025 huggingface dataset:
The stability of the RNA bases: Implications for the origin of life
Authors: Levy Matthew , Miller Stanley L.
Published: July 2002

High-temperature origin-of-life theories require that the components of the first genetic material are stable. We therefore have measured the half-lives for the decomposition of the nucleobases. They have been found to be short on the geologic time scale. At 100°C, the growth temperatures of the hyperthermophiles, the half-lives are too short to allow for the adequate accumulation of these compounds (
t
1/2
for A and G ≈ 1 yr; U = 12 yr; C = 19 days). Therefore, unless the origin of life took place extremely rapidly (<100 yr), we conclude that a high-temperature origin of life may be possible, but it cannot involve adenine, uracil, guanine, or cytosine. The rates of hydrolysis at 100°C also suggest that an ocean-boiling asteroid impact would reset the prebiotic clock, requiring prebiotic synthetic processes to begin again. At 0°C, A, U, G, and T appear to be sufficiently stable (
t
1/2
≥ 10
6
yr) to be involved in a low-temperature origin of life. However, the lack of stability of cytosine at 0°C (
t
1/2
= 17,000 yr) raises the possibility that the GC base pair may not have been used in the first genetic material unless life arose quickly (<10
6
yr) after a sterilization event. A two-letter code or an alternative base pair may have been used instead.
Leukoregulin induction of protein expression in human orbital fibroblasts: Evidence for anatomical site-restricted cytokine–target cell interactions
Authors: Young Donald A. , Evans Charles H. , Smith Terry J.
Published: July 2002

The molecular basis for the profound inflammatory response and the accumulation of hyaluronan in orbital connective tissues seen in thyroid-associated ophthalmopathy is unknown. Moreover, the link between the orbital manifestations of Graves’ disease and those in the pretibial skin, localized dermopathy, has yet to be established. We have reported recently that leukoregulin, an activated T lymphocyte-derived cytokine, dramatically induces hyaluronan synthesis and prostaglandin–endoperoxide H synthase 2 in human orbital fibroblasts in culture. In the current studies, utilizing giant two-dimensional gel electrophoresis, we find that orbital fibroblasts express constitutively a protein profile that distinguishes them from skin fibroblasts derived from the abdominal wall and from the pretibium. We further demonstrate that leukoregulin, when present in culture medium for 16 hr, up-regulates a set of orbital fibroblast proteins not present in untreated cultures or in fibroblasts from the abdominal wall. However, some of the same protein inductions are present in the pretibial fibroblasts. These leukoregulin-induced changes in protein expression are completely blocked by dexamethasone (10 nM). Our findings are the first to identify proteins that appear to be expressed and differentially regulated in an anatomical site-restricted manner in orbital and pretibial fibroblasts and seem to establish a molecular link between fibroblasts from the orbit and those in pretibial skin.
Conformation gating as a mechanism for enzyme specificity
Authors: Zhou Huan-Xiang , Wlodek Stanislaw T. , McCammon J. Andrew
Published: July 2002

Acetylcholinesterase, with an active site located at the bottom of a narrow and deep gorge, provides a striking example of enzymes with buried active sites. Recent molecular dynamics simulations showed that reorientation of five aromatic rings leads to rapid opening and closing of the gate to the active site. In the present study the molecular dynamics trajectory is used to quantitatively analyze the effect of the gate on the substrate binding rate constant. For a 2.4-Å probe modeling acetylcholine, the gate is open only 2.4% of the time, but the quantitative analysis reveals that the substrate binding rate is slowed by merely a factor of 2. We rationalize this result by noting that the substrate, by virtue of Brownian motion, will make repeated attempts to enter the gate each time it is near the gate. If the gate is rapidly switching between the open and closed states, one of these attempts will coincide with an open state, and then the substrate succeeds in entering the gate. However, there is a limit on the extent to which rapid gating dynamics can compensate for the small equilibrium probability of the open state. Thus the gate is effective in reducing the binding rate for a ligand 0.4 Å bulkier by three orders of magnitude. This relationship suggests a mechanism for achieving enzyme specificity without sacrificing efficiency.
The leukotriene B4receptor functions as a novel type of coreceptor mediating entry of primary HIV-1 isolates into CD4-positive cells
Authors: Owman Christer , Garzino-Demo Alfredo , Cocchi Fiorenza , Popovic Mikulas , Sabirsh Alan , Gallo Robert C.
Published: July 2002

The recently cloned human chemoattractant receptor-like (CMKRL)1, which is expressedin vivoin CD4-positive immune cells, has structural homology with the two chemokine receptors C-C chemokine receptor (CCR)5 and C-X-C chemokine receptor (CXCR)4, which serve as the major coreceptors necessary for fusion of the HIV-1 envelope with target cells. In view of the structural similarity, CMKRL1 was tested for its possible function as another HIV-1 coreceptor after stable expression in murine fibroblasts bearing the human CD4 receptor. The cells were infected with 10 primary clinical isolates of HIV-1, and entry was monitored by semiquantitative PCR of viral DNA. The efficiency of the entry was compared with the entry taking place in CD4-positive cells expressing either CCR5 or CXCR4. Seven of the isolates used CMKRL1 for viral entry; they were mainly of the syncytium-inducing phenotype and also used CXCR4. Entry efficiency was higher with CMKRL1 than with CXCR4 for more than half of these isolates. Three of the ten isolates did not use CMKRL1; instead, entry was mediated by both CCR5 and CXCR4. The experiments thus indicate that CMKRL1 functions as a coreceptor for the entry of HIV-1 into CD4-positive cells. In the course of this study, leukotriene B4was shown to be the natural ligand for this receptor (now designated BLTR), which therefore represents a novel type of HIV-1 coreceptor along with the previously identified chemokine receptors. BLTR belongs to the same general chemoattractant receptor family as the chemokine receptors but is structurally more distant from them than are any of the previously described HIV-1 coreceptors.
The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface
Authors: Bals Robert , Wang Xiaorong , Zasloff Michael , Wilson James M.
Published: July 2002

The airway surface is an important host defense against pulmonary infection. Secretion of proteins with antimicrobial activity from epithelial cells onto the airway surface represents an important component of this innate immune system. Defensins are the best characterized epithelial-derived peptide antibiotics. A member of another family of peptide antibiotics called cathelicidins recently was identified from human bone marrow. We show in this paper that this human peptide named LL-37/hCAP-18 also may play a role in innate immunity of the human lung.In situhybridization localized high levels of LL-37/hCAP-18 RNA to surface epithelial cells of the conducting airway as well as serous and mucous cells of the submucosal glands. LL-37/hCAP-18 peptide with antimicrobial activity was partially purified from airway surface fluid from human lung and a human bronchial xenograft model. The synthetic peptide LL-37 demonstrated antibiotic activity against a number of Gram-negative and Gram-positive organisms includingPseudomonas aeruginosa; bacterial killing of LL-37 was sensitive to NaCl and was synergistic with lactoferrin and lysozyme. In summary, we show that LL-37/hCAP-18 is a peptide with broad antimicrobial activity that is secreted onto the airway surface from epithelial cells of the human lung.
The 16-kDa α-crystallin (Acr) protein ofMycobacterium tuberculosisis required for growth in macrophages
Authors: Yuan Ying , Crane Deborah D. , Simpson R. Mark , Zhu YaQi , Hickey Mark J. , Sherman David R. , Barry Clifton E.
Published: July 2002

Although the 16-kDa α-crystallin homologue ofMycobacterium tuberculosis(MTB) is the dominant protein produced by stationary phase culturesin vitro, it is undetectable in logarithmically growing cultures. By growing bacilli at defined oxygen concentrations,acrtranscription was shown to be strongly induced by mildly hypoxic conditions. Acr expression also was found to be induced during the course ofin vitroinfection of macrophages. Theacrgene was replaced with a hygromycin resistance cassette by allelic exchange in MTB H37Rv. The resulting Δacr∷hptstrain was shown to be equivalent to wild-type H37Rv inin vitrogrowth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells. In addition to its proposed role in maintenance of long-term viability during latent, asymptomatic infections, these results establish a role for the Acr protein in replication during initial MTB infection.
A novel frizzled gene identified in human esophageal carcinoma mediates APC/β-catenin signals
Authors: Tanaka Shinji , Akiyoshi Tsuyoshi , Mori Masaki , Wands Jack R. , Sugimachi Keizo
Published: July 2002

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis revealed that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and β-catenin followed by nuclear translocation of β-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibited the interaction of APC with β-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhanced β-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance β-catenin mediated signals in poorly differentiated human esophageal carcinomas.
Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin
Authors: Kumar Challa V. , Buranaprapuk Apinya , Opiteck Gregory J. , Moyer Mary B. , Jockusch Steffen , Turro Nicholas J.
Published: July 2002

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by
N-
(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 10
5
M
−1
and 6.5 ± 0.4 × 10
7
M
−1
, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at ≈460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.
Tn
5
/IS
50
target recognition
Authors: Goryshin Igor Y. , Miller Joanna A. , Kil Yuri V. , Lanzov Vladislav A. , Reznikoff William S.
Published: July 2002

This communication reports an analysis of Tn
5
/IS
50
target site selection by using an extensive collection of Tn
5
and IS
50
insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from
in vitro
and
in vivo
transposition events. Since the data sets are consistent and transposase was the only protein present
in vitro
, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn
5
/IS
50
bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn
5
/IS
50
target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn
5
/IS
50
target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.
The plant cDNA
LCT1
mediates the uptake of calcium and cadmium in yeast
Authors: Clemens Stephan , Antosiewicz Danuta M. , Ward John M. , Schachtman Daniel P. , Schroeder Julian I.
Published: July 2002

Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd
2+
transport we tested putative plant cation transporters for Cd
2+
uptake activity by expressing cDNAs in
Saccharomyces cerevisiae
and found that expression of one clone,
LCT1
, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd
2+
sensitivity is correlated with an increase in Cd
2+
uptake.
LCT1
-dependent Cd
2+
uptake is saturable, lies in the high-affinity range (apparent
K
M
for Cd
2+
= 33 μM) and is sensitive to block by La
3+
and Ca
2+
. Growth assays demonstrated a sensitivity of
LCT1
-expressing yeast cells to extracellular millimolar Ca
2+
concentrations.
LCT1
-dependent increase in Ca
2+
uptake correlated with the observed phenotype. Furthermore,
LCT1
complements a yeast disruption mutant in the
MID1
gene, a non-
LCT1
-homologous yeast gene encoding a membrane Ca
2+
influx system required for recovery from the mating response. We conclude that
LCT1
mediates the uptake of Ca
2+
and Cd
2+
in yeast and may therefore represent a first plant cDNA encoding a plant Ca
2+
uptake or an organellar Ca
2+
transport pathway in plants and may contribute to transport of the toxic metal Cd
2+
across plant membranes.
Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules
Authors: Steinle Alexander , Groh Veronika , Spies Thomas
Published: July 2002

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.
BRCA1 is associated with the centrosome during mitosis
Authors: Hsu Lih-Ching , White Raymond L.
Published: July 2002

Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. The centrosome replicates during the cell cycle, directs the assembly of bipolar mitotic spindles, and plays an important role in maintaining the fidelity of cell division. Recently, tumor suppressors such as p53 and retinoblastoma protein pRB have been localized to the centrosome in a cell cycle-dependent manner. Immunofluorescence microscopy and analysis of isolated centrosomes now provide evidence that BRCA1 protein, a suppressor of tumorigenesis in breast and ovary, also is associated with centrosomes during mitosis. Our results indicate that BRCA1 localizes with the centrosome during mitosis and coimmunoprecipitates with γ-tubulin, a centrosomal component essential for nucleation of microtubules. Furthermore, γ-tubulin associates preferentially with a hypophosphorylated form of BRCA1.
The lipid phosphatase activity of PTEN is critical for its tumor supressor function
Authors: Myers Michael P. , Pass Ian , Batty Ian H. , Van der Kaay Jeroen , Stolarov Javor P. , Hemmings Brian A. , Wigler Michael H. , Downes C. Peter , Tonks Nicholas K.
Published: July 2002

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.
Interaction between like-charged colloidal spheres in electrolyte solutions
Authors: Wu Jianzhong , Bratko Dusan , Prausnitz John M.
Published: July 2002

How colloidal particles interact with each other is one of the key issues that determines our ability to interpret experimental results for phase transitions in colloidal dispersions and our ability to apply colloid science to various industrial processes. The long-accepted theories for answering this question have been challenged by results from recent experiments. Herein we show from Monte-Carlo simulations that there is a short-range attractive force between identical macroions in electrolyte solutions containing divalent counterions. Complementing some recent and related results by others, we present strong evidence of attraction between a pair of spherical macroions in the presence of added salt ions for the conditions where the interacting macroion pair is not affected by any other macroions that may be in the solution. This attractive force follows from the internal-energy contribution of counterion mediation. Contrary to conventional expectations, for charged macroions in an electrolyte solution, the entropic force is repulsive at most solution conditions because of localization of small ions in the vicinity of macroions. Both Derjaguin–Landau–Verwey–Overbeek theory and Sogami–Ise theory fail to describe the attractive interactions found in our simulations; the former predicts only repulsive interaction and the latter predicts a long-range attraction that is too weak and occurs at macroion separations that are too large. Our simulations provide fundamental “data” toward an improved theory for the potential of mean force as required for optimum design of new materials including those containing nanoparticles.
Nativelike topology assembly of small proteins using predicted restraints in Monte Carlo folding simulations
Authors: Ortiz Angel R. , Kolinski Andrzej , Skolnick Jeffrey
Published: July 2002

By incorporating predicted secondary and tertiary restraints derived from multiple sequence alignments into
ab initio
folding simulations, it has been possible to assemble native-like tertiary structures for a test set of 19 nonhomologous proteins ranging from 29 to 100 residues in length and representing all secondary structural classes. Secondary structural restraints are provided by the PHD secondary structure prediction algorithm that incorporates multiple sequence information. Multiple sequence alignments also provide predicted tertiary restraints via a two-step process: First,
seed
side chain contacts are selected from a correlated mutation analysis, and then an inverse folding algorithm expands these seed contacts. The predicted secondary and tertiary restraints are incorporated into a lattice-based, reduced protein model for structure assembly and refinement. The resulting native-like topologies exhibit a coordinate root—mean–square deviation from native for the whole chain between 3.1 and 6.7 Å, with values ranging from 2.6 to 4.1 Å over ≈80% of the structure. Overall, this study suggests that the use of restraints derived from multiple sequence alignments combined with a fold assembly algorithm is a promising approach to the prediction of the global topology of small proteins.
Fine structure of neural spiking and synchronization in the presence of conduction delays
Authors: Ermentrout G. B. , Kopell N.
Published: July 2002

Hippocampal networks of excitatory and inhibitory neurons that produce γ-frequency rhythms display behavior in which the inhibitory cells produce spike doublets when there is strong stimulation at separated sites. It has been suggested that the doublets play a key role in the ability to synchronize over a distance. Here we analyze the mechanisms by which timing in the spike doublet can affect the synchronization process. The analysis describes two independent effects: one comes from the timing of excitation from separated local circuits to an inhibitory cell, and the other comes from the timing of inhibition from separated local circuits to an excitatory cell. We show that a network with both of these effects has different synchronization properties than a network with either excitatory or inhibitory type of coupling alone, and we give a rationale for the shorter space scales associated with inhibitory interactions.
Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia
Authors: Boas Franz Edward , Forman Linda , Beutler Ernest
Published: July 2002

Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we usedN-hydroxysuccinimide-biotin to tag rabbit red cellsin vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells.
Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors
Authors: Li Ke-Jun , Garoff Henrik
Published: July 2002

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to produce retrovirus vectors that can transduce and integrate genes with introns. However, this has proven difficult because the viral RNA is spliced when it is synthesized in the nucleus of a producer cell. We describe a novel approach to avoid this problem. In our system the retroviral RNA is synthesized in the cytoplasm of the cell, not in the nucleus, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leukemia virus genome containing the chloramphenicol acetyltransferase (CAT) gene in association with an intron. This was inserted into a SFV transcription plasmid and the corresponding SFV vector RNA was transcribed
in vitro
. BHK-21 cells were then transfected with this vector RNA together with two additional SFV vectors that encode the Moloney murine leukemia virus packaging proteins. Retrovirus vectors containing intron-CAT sequences were produced at titers up to 1.3 × 10
6
infectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into NIH 3T3 cells, where the intron-CAT RNA was subjected to efficient splicing and used for high level enzyme expression. Thus, the results show that intron containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.
Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells
Authors: Kuge Osamu , Hasegawa Kazuhide , Saito Kyoko , Nishijima Masahiro
Published: July 2002

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of
l
-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene,
pssA
, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant
pssA
cDNA, but not the wild-type
pssA
cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type
pssA
-transfected cells was inhibited by PtdSer, but that of the mutant
pssA
-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant
pssA
-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type
pssA
-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.
Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells
Authors: Rescigno Maria , Citterio Stefania , Thèry Clotilde , Rittig Michael , Medaglini Donata , Pozzi Gianni , Amigorena Sebastian , Ricciardi-Castagnoli Paola
Published: July 2002

Here, we show that bacteria inducede novosynthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinantStreptococcus gordoniiis processed and presented on MHC class I molecules. This presentation is 106times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.
The hypoglossal canal and the origin of human vocal behavior
Authors: Kay Richard F. , Cartmill Matt , Balow Michelle
Published: July 2002

The mammalian hypoglossal canal transmits the nerve that supplies the muscles of the tongue. This canal is absolutely and relatively larger in modern humans than it is in the African apes (
Pan
and
Gorilla
). We hypothesize that the human tongue is supplied more richly with motor nerves than are those of living apes and propose that canal size in fossil hominids may provide an indication about the motor coordination of the tongue and reflect the evolution of speech and language. Canals of gracile
Australopithecus
, and possibly
Homo habilis
, fall within the range of extant
Pan
and are significantly smaller than those of modern
Homo
. The canals of Neanderthals and an early “modern”
Homo sapiens
(Skhul 5), as well as of African and European middle Pleistocene
Homo
(Kabwe and Swanscombe), fall within the range of extant
Homo
and are significantly larger than those of
Pan troglodytes
. These anatomical findings suggest that the vocal capabilities of Neanderthals were the same as those of humans today. Furthermore, the vocal abilities of
Australopithecus
were not advanced significantly over those of chimpanzees whereas those of
Homo
may have been essentially modern by at least 400,000 years ago. Thus, human vocal abilities may have appeared much earlier in time than the first archaeological evidence for symbolic behavior.
A two-step, two-signal model for the primary activation of precursor helper T cells
Authors: Bretscher Peter A.
Published: July 2002

I present here a new model for the primary activation of precursor helper T cells. Observations demonstrate that the immune system learns not to respond to extrathymic, organ-specific self-antigens because of their early appearance in development. The immune system thus discriminates between peripheral self-antigens and foreign antigens and, when mature, usually makes an immune response against only the latter. Contemporary models for the activation and inactivation of T helper (Th) function do not account for such discrimination. The model proposed here is consistent with contemporary findings and incorporates a mechanism of peripheral self–nonself discrimination.
The
Lateral suppressor (Ls)
gene of tomato encodes a new member of the VHIID protein family
Authors: Schumacher Karin , Schmitt Thomas , Rossberg Mathias , Schmitz Gregor , Theres Klaus
Published: July 2002

The ability of the shoot apical meristem to multiply and distribute its meristematic potential through the formation of axillary meristems is essential for the diversity of forms and growth habits of higher plants. In the
lateral suppressor
mutant of tomato the initiation of axillary meristems is prevented, thus offering the unique opportunity to study the molecular mechanisms underlying this important function of the shoot apical meristem. We report here the isolation of the
Lateral suppressor
gene by positional cloning and show that the mutant phenotype is caused by a complete loss of function of a new member of the VHIID family of plant regulatory proteins.
Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus
Authors: Zanta Maria Antonietta , Belguise-Valladier Pascale , Behr Jean-Paul
Published: July 2002

Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of the cationic vector or the cell type used. A lysine to threonine mutation of the third NLS amino acid completely abolished these remarkable features, suggesting importin-mediated translocation. Our hypothesis is that the 3-nm-wide DNA present in the cytoplasm is initially docked to and translocated through a nuclear pore by the nuclear import machinery. As DNA enters the nucleus, it is quickly condensed into a chromatin-like structure, which provides a mechanism for threading the remaining worm-like molecule through the pore. A single NLS signal is thus sufficient, whereas many signals on a gene would actually inhibit entry, the same DNA molecule being threaded through adjacent pores.
Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11
Authors: Snow Bryan E. , Betts Laurie , Mangion Joan , Sondek John , Siderovski David P.
Published: July 2002

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to G
β
5
subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human
RGS6
. When RGS6 is coexpressed with different G
β
subunits, only RGS6 and G
β
5
interact. The expression of mRNA for
RGS6
and
G
β
5
in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of G
β
binding by GGL domain mutants, support the contention that G
γ
-like regions within RGS proteins interact with G
β
5
subunits in a fashion comparable to conventional G
β
/G
γ
pairings. Mutation of the highly conserved Phe-61 residue of G
γ
2
to tryptophan, the residue present in all GGL domains, increases the stability of the G
β
5
/G
γ
2
heterodimer, highlighting the importance of this residue to GGL/G
β
5
association.
A set of independent selectable markers for transfection of the human malaria parasite
Plasmodium falciparum
Authors: Mamoun Choukri Ben , Gluzman Ilya Y. , Goyard Sophie , Beverley Stephen M. , Goldberg Daniel E.
Published: July 2002

Genomic information is rapidly accumulating for the human malaria pathogen,
Plasmodium falciparum.
Our ability to perform genetic manipulations to understand
Plasmodium
gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of
P. falciparum
. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes
BSD
, encoding blasticidin S deaminase of
Aspergillus terreus
, and
NEO
, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (
HRP
III) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and
BSD
or
NEO
expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the
P. falciparum
genome.
EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology
Authors: Rong Yongqi , Doctrow Susan R. , Tocco Georges , Baudry Michel
Published: July 2002

The present study tested the effects of EUK-134, a synthetic superoxide dismutase/catalase mimetic, on several indices of oxidative stress and neuropathology produced in the rat limbic system as a result of seizure activity elicited by systemic kainic acid (KA) administration. Pretreatment of rats with EUK-134 did not modify the latency for or duration of KA-induced seizure activity. It did produce a highly significant reduction in increased protein nitration, activator protein-1- and NF-κB-binding activity, and spectrin proteolysis as well as in neuronal damage resulting from seizure activity in limbic structures. These results support the hypothesis that kainate-induced excitotoxicity is caused, at least in part, by the action of reactive oxygen species. Furthermore, they suggest that synthetic superoxide dismutase/catalase mimetics such as EUK-134 might be used to prevent excitotoxic neuronal injury.
Origins, colonization, and lineage recombination in a widespread perennial soybean polyploid complex
Authors: Doyle J. J. , Doyle J. L. , Brown A. H. D.
Published: July 2002

Polyploidy is a dominant feature of flowering plant genomes, including those of many important crop species, implying that polyploidy confers evolutionary advantages on plant species. Recent molecular studies suggest that polyploids often originate many times from the same progenitor diploids. For this to provide a broader genetic base for a polyploid species, there must be lineage recombination in the genomes of polyploids having different origins, and this has rarely been documented in recently formed wild polyploid species.
Glycine tabacina
, a wild relative of soybean, forms a widespread polyploid complex in Australia and the islands of the Pacific Ocean. In a sample of 40
G. tabacina
plants, DNA sequence variation at one homoeologous histone H3-D locus identified three alleles, each also found in Australian diploid
Glycine
species. These data agree with our previous studies of chloroplast DNA variation in suggesting that this polyploid has originated several times. Both the origins of the polyploid and several independent dispersals from Australia to oceanic islands appear to have occurred within the last 30,000 years. The distributions of histone alleles, chloroplast haplotypes, and alleles at two isozyme loci were uncorrelated, and 20 multilocus genotypes were found among the 40 plants sampled. Extensive lineage recombination is thus hypothesized in the polyploid, involving migration and occasional outcrossing in this predominantly inbreeding species. The combination of multiple origins with gene exchange among lineages increases the genetic base of a polyploid and may help explain the wide colonization of polyploid
G. tabacina
relative to its diploid progenitors.
Prevention of interleukin-8-induced mobilization of hematopoietic progenitor cells in rhesus monkeys by inhibitory antibodies against the Metalloproteinase gelatinase B (MMP-9)
Authors: Pruijt Johannes F. M. , Fibbe Willem E. , Laterveer Laurens , Pieters Reagan A. , Lindley Ivan J. D. , Paemen Liesbet , Masure Stefan , Willemze Roel , Opdenakker Ghislain
Published: July 2002

Previously, we demonstrated that IL-8 induces rapid mobilization of hematopoietic progenitor cells (HPC) from the bone marrow of rhesus monkeys. Because activation of neutrophils by IL-8 induces the release of gelatinase B (MMP-9), which is involved in the degradation of extracellular matrix molecules, we hypothesized that MMP-9 release might induce stem cell mobilization by cleaving matrix molecules to which stem cells are attached. Rhesus monkeys were treated with a single i.v. injection of 0.1 mg/kg human IL-8, which resulted in a 10- to 100-fold increase in HPC within 30 min after injection. Zymographic analysis revealed a dramatic instantaneous increase in the plasma levels of MMP-9, followed by the increase in circulating HPC. Enzyme levels decreased at 2 h after injection of IL-8, simultaneously with the decrease in the numbers of circulating HPC. To test the hypothesis that MMP-9 induction was involved in HPC mobilization, rhesus monkeys were treated with a highly specific inhibitory monoclonal anti-gelatinase B antibody. Anti-gelatinase B at a dose of 1–2 mg/kg completely prevented the IL-8-induced mobilization of HPC, whereas a dose of 0.1 mg/kg had only a limited effect. Preinjection of inhibitory antibodies did not preclude the IL-8-induced production and secretion of MMP-9. Pretreatment with an irrelevant control antibody did not affect IL-8-induced mobilization, showing that the inhibition by the anti-gelatinase B antibody was specific. In summary, IL-8 induces the rapid systemic release of MMP-9 with concurrent mobilization of HPC that is prevented by pretreatment with an inhibitory anti-gelatinase B antibody, indicating that MMP-9 is involved as a mediator of the IL-8-induced mobilization of HPC.
High pressure fosters protein refolding from aggregates at high concentrations
Authors: St. John Richard J. , Carpenter John F. , Randolph Theodore W.
Published: July 2002

High hydrostatic pressures (1–2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and β-lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml. Inclusion bodies containing β-lactamase could be refolded at high yields of active protein, even without added GdmHCl.
Emergence ofFY*Anullin aPlasmodium vivax-endemic region of Papua New Guinea
Authors: Zimmerman Peter A. , Woolley Ian , Masinde Godfred L. , Miller Stephanie M. , McNamara David T. , Hazlett Fred , Mgone Charles S. , Alpers Michael P. , Genton Blaise , Boatin B. A. , Kazura James W.
Published: July 2002

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is α+-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that α+-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that α+-thalassemia may facilitate so-called “benign”Plasmodium vivaxinfection to act later in life as a “natural vaccine” against severePlasmodium falciparummalaria. Here, in aP. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of α+-thalassemia ≥0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a−b−]) on theFY*Aallele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*Anull) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence ofP. vivaxinfection inFY*A/FY*A(83/508 = 0.163) compared withFY*A/FY*Anull(2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47–8.91). Emergence ofFY*Anullin this population suggests thatP. vivaxis involved in selection of this erythroid polymorphism. This mutation would ultimately compromise α+-thalassemia/P. vivax-mediated protection against severeP. falciparummalaria.
Mechanism of biological synergy between cellular Src and epidermal growth factor receptor
Authors: Tice David A. , Biscardi Jacqueline S. , Nickles Amanda L. , Parsons Sarah J.
Published: July 2002

Overexpression of both cellular Src (c-Src) and the epidermal
growth factor receptor (EGFR) occurs in many of the same human tumors,
suggesting that they may functionally interact and contribute to the
progression of cancer. Indeed, in murine fibroblasts, overexpression of
c-Src has been shown to potentiate the mitogenic and tumorigenic
capacity of the overexpressed EGFR. Potentiation correlated with the
ability of c-Src to physically associate with the activated EGFR and
the appearance of two unique
in vivo
phosphorylations on
the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of
C3H10T½ murine fibroblasts that contain kinase-deficient (K−)
c-Src and overexpressed wild-type EGFR, we show that the kinase
activity of c-Src is required for both the biological synergy with the
receptor and the phosphorylations on the receptor, but not for the
association of c-Src with the receptor. In transient transfection
assays, not only epidermal growth factor but also serum- and
lysophosphatidic acid-induced DNA synthesis was ablated in a
dominant-negative fashion by a Y845F mutant of the EGFR, indicating
that c-Src-induced phosphorylation of Y845 is critical for the
mitogenic response to both the EGFR and a G protein-coupled receptor
(lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR
was found to retain its full kinase activity and its ability to
activate the adapter protein SHC and extracellular signal-regulated
kinase ERK2 in response to EGF, demonstrating that the mitogenic
pathway involving phosphorylation of Y845 is independent of
ERK2-activation. The application of these findings to the development
of novel therapeutics for human cancers that overexpress c-Src and EGFR
is discussed.
The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure
Authors: Hamm Peter , Lim Manho , DeGrado William F. , Hochstrasser Robin M.
Published: July 2002

A form of two-dimensional (2D) vibrational spectroscopy, which uses
two ultrafast IR laser pulses, is used to examine the structure of a
cyclic penta-peptide in solution. Spectrally resolved cross peaks occur
in the off-diagonal region of the 2D IR spectrum of the amide I region,
analogous to those in 2D NMR spectroscopy. These cross peaks measure
the coupling between the different amide groups in the structure. Their
intensities and polarizations relate directly to the three-dimensional
structure of the peptide. With the help of a model coupling
Hamiltonian, supplemented by density functional calculations, the
spectra of this penta-peptide can be regenerated from the known
solution phase structure. This 2D-IR measurement, with an intrinsic
time resolution of less than 1 ps, could be used in all time regimes of
interest in biology.